A Practical Manual for Instrumentation

A Practical Manual

of

Instrumentation

For

Students of M.Sc. Microbiology

(Kantipur College of Medical Sciences)

Prepared by:

Upendra Thapa Shrestha

Faculty

Kantipur College of Medical Sciences

Department of Microbiology

Tribhuvan University

Sitapaila, <Ș>Kathmandu

2010

ACKNOWLEDGEMENT

It gives me immense pleasure to express my heartfelt appreciation to all the people who helped me in one way or another to complete this practical instrumentation manual.

Respectfully, I would like to express my sincere gratitude to Kantipur College of Medical Sciences (KCMS), Sitapaila, Kathmandu and all family of college for considering this manual suitable for M. Sc Microbiology program and publishing it.

I am indebted to my colleagues Mr. Nawaraj Adhikari, a coordinator, KCMS and Mr. Dhiraj Acharya, a faculty member of KCMS for supporting to publish.

I am much obliged to Mr. Kiran Babu Tiwari, a PhD scholar and Faculty member of KCMS for helping me to prepare this manual.

Finally I would like to appreciate my cousins Sagar and Saurav for helping me on typing this manual.

Lists of Experiments:

Experiment no 1: Introduction of different instruments used in Biochemistry and Instrumentation practical lab.

Experiment no 2: Preparation of different types of solutions (Percent, Molar, Normal, Solutions from Solutions and Titrations, Concentrated Solutions Saturated) used in biochemistry lab.

Experiment no 3: Preparation of different types of Buffers (Phosphate, Acetate, Citrate, Tris buffers, etc) used in biochemical studies.

Experiment no 4: Preparation of various sub-cellular fractions of liver cells (rat, chicken…) by Differential centrifugation method

Experiment no 5: Fractionation of serum protein by salt (Sodium chloride and Ammonium sulphate=separation of albumin and globulin)

Experiment no 6: Precipitation and separation of protein of interest by TCA (Trichloroacetic acid) method from biological sample.

Experiment no 7: Separation and identification of amino acids in a given mixture by ascending paper chromatography.

Experiment no 8: Separation and identification of amino acids in a given mixture by two dimensional paper chromatography.

Experiment no 9: Separation and identification of sugars (sugar juices) by adsorption Thin layer chromatography (TLC).

Experiment no 10: Extraction and identification of lipids from given biological samples (egg, cooking oils…..) by Thin layer chromatography.

Experiment no 11: Purification of bovine serum albumin from buffalo serum by size exclusion chromatography (gel filtration)

Experiment no 12: Determination of molecular weight of a given protein by SDS-PAGE (Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis).

Experiment no 13: Native Disc gel electrophoresis of proteins.

Experiment no 14: Determination of molecular weight of DNA (genomic and plasmid)by Agarose gel electrophoresis.

Experiment no 15: Determination of l_max of different colored and noncolored compounds by spectrophotometer.

Experiment no 16: Verification of validity of Beer’s law and determination of the molar extinction coefficient of BSA

Experiment no. 1

Introduction of different instruments used in Biochemistry and Instrumentation practical laboratory

Lists of instruments used in lab:

1. Micropipettes

a.

b.

c.

d.

e.

2. Spectrophotometer

a.

b.

c.

d.

e.

3. UV illuminator

a.

b.

c.

d.

e.

4. Centrifuge

a.

b.

c.

d.

e.

5. Homogenizer

a.

b.

c.

d.

e.

6. SDS-set

a.

b.

c.

d.

e.

7. Horizontal Electrophoresis set

a.

b.

c.

d.

e.

8. Others……

Experiment no. 2

Preparation of different types of solutions (Percent, Molar, Normal, Solutions from Solutions and Titrations, Concentrated Solutions Saturated) used in biochemistry lab

Objectives:

1. To prepare Percent, Molar, Normal, Solutions from Solutions and Titrations, Concentrated Solutions and Saturated solutions.

Principle:

1. Percent solutions

There are three types of percent solutions. All are parts of solute per 100 total parts of solution.

Based on the following definitions you may calculate the concentration of a solution or calculate how to make up a specific concentration.

1. % W/W - Percent of weight of solute in the total weight of the solution. Percent here is the number of grams of solute in 100 grams of solution.

Example:

A 100% (W/W) NaCl solution is made by weighing 100 g NaCl and dissolving in 100 g of solution.

2. % W/V - Percent of weight of solution in the total volume of solution. Percent here is the number of grams of solute in 100 ml of solution. This is probably the least significant way of naming a solution, but the most common way of doing it. In fact, any percent solution not stipulated as W/W, W/V, or V/V is assumed to be % W/V.

Example:

A 4% (W/V) NaCl solution is 4 g of NaCl in 100 ml of solution.

3. % V/V - Percent of volume of solute in the total volume of solution %V/V. Percent here is the number of milliliters of solute in 100 ml of solution.

Example:

A 10% (V/V) ethanol solution is 10 ml of ethanol in 100 ml of solution; unless otherwise stated, water is the solvent.

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MOLAR SOLUTIONS (M)

The definition of molar solution is a solution that contains 1 mole of solute in each liter of solution. A mole is the number of gram molecular weights (gmw). Therefore, we can also say a 1M = 1 gMW solute/liter solution.

1M NaCl solution would be

Na = MW of 23

Cl = MW of 35.5

NaCl = MW of 58.5

1M = 58.5 g of NaCl in 1 liter of solution.

It may be made by weighing out 58.5 g of NaCl and qs to 1 liter with water. The qs stands for quality sufficient and is a term used to designate that the total volume must be 1 liter (or whatever is stated).

58.5 g NaCl qs 1 liter with H20

Examples of other solutions would be

1 M H₂S0₄ = 98 g/L

1 M H₃P0₄ = 98 g/L

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NORMAL SOLUTIONS:

The definition of a normal solution is a solution that contains 1 gram equivalent weight (gEW) per liter solution. An equivalent weight is equal to the molecular weight divided by the valence (replaceable H ions).

1N NaCl = 58.5 g/L

1N HCl = 36.5 g/L

1N H₂S0₄ = 49 g/L

Problems involving normality are worked the same as those involving molarity but the valence must be considered:

1N HCl the MW= 36.5 the EW = 36.5 and 1N would be 36.5 g/L

1N H₂SO₄ the MW = 98 the EW = 49 and 1N would be 49 g/L

1N H₃PO₄ the MW = 98 the EW = 32.7 and 1N would be 32.7 g/L

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SOLUTIONS FROM SOLUTIONS AND TITRATIONS:

Many times the solutions we make are made from more concentrated solutions rather than dry chemicals. For figuring these out it can just be easier to remember a formula than figuring them out.

That formula is:

V₁C₁ = V₂C₂

Where V = volume

C = concentration (%, M, N)

1 = the more concentrated solution

2 = the new (dilute) solution

or in other words the volume of a concentrated solution times its concentration will contain the proper amount of chemical to give the volume of a weaker solution times its concentration.

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CONCENTRATED SOLUTIONS:

Concentrated acids and bases and other stock reagents exist as liquids and usually do not have their concentrations listed as %, M or N. They usually have specific gravity or density of the solution. The concentration may be calculated from this. Then how to make the weaker solution from this concentration solution is determined. So when the only information about the concentration of a concentrated solution is specific gravity and percent assay, we must first calculate concentration.

Specific Gravity for all purposes is the number of grams per milliliter.

For example:

HCl sp. gr. = 1.080 would mean that there is 1.080 g of HCl in every ml of solution.

Since N is in gEW per liter we need to convert to liter.

1.080 g/ml = 1080 g/L

If 1 N = 36.5 g/L

Then 1080 g/L / 36.5 g/L = 29.6N

One problem with most purchased solutions, even the best, is that they are not pure. Some of that weight is due to other substances. But the bottle will state the percent assay or what percent is really there.

e.g., 95% HCl would mean that 95% of 1.080 is HCl.

In that case

(1080) (0.95) = 1026 g/L

Now that we know the amount of solute per volume of solution, we may calculate concentration.

If we want to know concentration in % (W/V), we say (as in previous problems)

1026g / 1L is the same as 1026g /1000 ml

1026g /1000 ml = X / 100 ml

X = 102.6 g

If we have 102.6 g/100 ml that is 102.6%.

If we had wanted to know concentration as molarity we would proceed as with other molarity problems:

1 M HCl = 36.5 g/L

1M / 36.5 g/L = X / 1026 g/L

28.1 M = X

Of course, your problem may be more than just determining concentration. You may need to make up 5 liters of 0.02N H₂S0₄ from concentrated H₂S0₄ on the shelf. The bottle states:

sp. gr. = 1.64

% assay = 80%

You don't give up and leave; you remember what you did in your lab math program -- which was:

First calculate the concentration of the H₂S0₄ in the bottle.

Sp. gr. = 1.64

= 1.64 g/ml

= 1640 g/L

% assay = 80%

Therefore, 1640 x 0.80 = 1312 g/L of H₂S0₄

MW H₂S0₄ = 98

E.W. = 49

1N H₂S0₄ = 49 g/L

1312 g/L / 49 g/L= 26.8N

Second, once you know the concentration, plug it into the formula.

V₁C₁ = V₂C₂

V₁ (26.8N) = (5,000 ml) (0.02N)

V₁ = (5000 ml x 0.02 N) / 26.8 N

V₁ = 3.7 ml

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SATURATED SOLUTIONS:

Often a procedure requires a saturated solution and does not stipulate an exact quantity that you need to weigh. The laboratory will have a Handbook of Physics and Chemistry or Chemistry Handbook which, among other information, lists the saturation index of compounds in water (and other solutions may be listed). Often this is listed as the number of grams of the chemical (solute) per 100 ml of solvent. For example, the solubility of KCl in cold water is 34.7. To make 100 ml of saturated KCl you would weigh >34.7g of KCl and qs to 100 ml.

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PART PER MILLIONS:

PPM is equivalent to mg/L

1 ppm=1 mg/L

SAMPLE PROBLEMS:

1. How much 12 N HCl do you need to make 400 ml of 2N solution?

2. You took 100 ml of a concentrated acid and made 2 liters of 0.5 N solutions. What was the normality of the original solution?

3. How much 7M H₂S0₄ will you need to make 100 ml of 7N H₂S0₄?

4. How much 0.2N H₂S0₄ may be made from 80 ml of 12N H₂S0₄?

5. You weigh out 80 g of NaOH pellets and dilute to 1 liter. What is the normality?

6. You weighed out 222g of CaCl₂ and diluted to 1 liter. What is the normality?

7. How would you make a liter of 4M CaCl₂?

8. How would you make 300 ml of a 0.5M NaOH solution?

9. You weighed out 58.5 g of NaCl and diluted it to 250 ml. What is the molarity of the solution?

Experiment no. 3

Preparation of different types of Buffers (Acetate, Phosphate, Tris buffers, Citrate-

phosphate.. etc) used in biochemical studies

Objectives:

1. To prepare the different buffers for biochemical studies.

Principle:

Solutions able to retain constant pH regardless of small amounts of acids or bases added are called buffers. Classical buffer contains solution of weak acid and conjugated base. Small amounts of acids or bases added are absorbed by buffer and the pH changes only slightly. In case of high or low pH just solutions of strong acids or bases are used - for example in case of pH=1 acid concentration is relatively high (0.1 M) and small addition of acid or base doesn't change pH of such solution significantly.

How to calculate pH of buffer solution containing both acid and conjugated base?

Dissociation constant definition can be rearranged into

or

(note that due to sign change [A^-] was moved to nominator).

This is so called Henderson-Hasselbalch equation (or buffer equation). It can be used for pH calculation of solution containing pair of acid and conjugate base - like HA/A^-, HA^-/A^2- or B⁺/BOH. For solutions of weak bases sometimes it s more convenient to use equation in the form

15.3

Both equations are perfectly equivalent and interchangeable.

Henderson-Hasselbalch equation is used mostly to calculate pH of solution created mixing known amount of acid and conjugate base (or neutralizing part of acid with strong base). For example, what is pH of solution prepared mixing reagents so that it contains 0.1 M of acetic acid and 0.05 M NaOH? Half of the acid is neutralized, so concentrations of acid and conjugate base are identical, thus quotient under logarithm is 1, logarithm is 0 and pH=pK_a.

This approach - while perfectly justifiable in many cases - is dangerous, as it creates false conviction that the equation can be used this way always. That's not true.

Henderson-Hasselbalch equation is valid when it contains equilibrium concentrations of acid and conjugate base. In case of solutions containing not-so-weak acids (or not-so-weak bases) equilibrium concentrations can be far from concentrations of substances put into solution.

Let's replace acetic acid from our example with something stronger - e.g. dichloroacetic acid, with pK_a=1.5. The same reasoning leads to result pH=1.5 - which is wrong. Proper pH value can be calculated from the equation 11.13 or using pH calculator - and it is 1.78. The reason is simple. Dichloroacetic acid is strong enough to dissociate on its own and equilibrium concentration of conjugate base is not 0.05 M (as we expected from the neutralization reaction stoichiometry) but 0.0334 M.

As a rule of thumb you may remember that acids with pK_a below 2.5 dissociate too easily and use of Henderson-Hasselbalch equation for pH prediction can give wrong results, especially in case of diluted solutions. For solutions above 10 mM and acids weaker than pK_a>=2.5, Henderson-Hasselbalch equation gives results with acceptable error. The same holds for bases with pK_b>=2.5. However, the same equation will work perfectly regardless of the pK_a value if you are asked to calculate ratio of acid to conjugated base in the solution with known pH.

Materials and Reagents:

1. pH meter

2. Glasswares-volumetric flasks, funnel

3. Filter papers

4. Phosphate Buffer: Monobasic sodium phosphate and Dibasic sodium phosphate

5. Acetate Buffer: Acetic acid and Sodium acetate

6. Citrate-phosphate Buffer: Citric acid and dibasic sodium phosphate

7. Tris Buffer: Tris (Hydroxymethl) aminomethane and HCl

Procedure:

1. Make stock solutions for Acetate Buffer.

A-0.2 M acetic acid (CH₃COOH)

B-0.2M sodium acetate (CH₃COONa)

X ml of A + y ml of B, dilute to a total volume of 100 ml

x	y	pH	x	y	pH
46.3	3.7	3.6	25.5	24.5	4.6
44.0	6.0	3.8	14.8	35.2	5.0
41.0	9.0	4.0	10.5	39.5	5.2
36.8	13.2	4.2	8.8	41.2	5.4
30.5	19.5	4.4	4.8	45.2	5.6

Adjust pH with pH meter by adding required component if necessary

2. Make stock solutions for Phosphate Buffer.

A-0.2 M monobasic sodium phosphate (NaH₂PO₄)

B-0.2M dibasic sodium phosphate (Na₂HPO₄)

X ml of A + y ml of B, dilute to a total volume of 200 ml

x	y	pH	x	y	pH
93.5	6.5	5.7	56.5	43.5	6.7
90.0	10.0	5.9	39.0	61.0	7.0
85.0	15.0	6.1	16.0	84.0	7.5
77.5	22.5	6.3	5.3	94.7	8.0
68.5	31.5	6.5

Adjust pH with pH meter by adding required component if necessary

3. Make stock solutions for Tris Buffer.

A-0.2 M Tris (hydroxymethly) aminomethane

B-0.2M HCl

X ml of A + y ml of B, dilute to a total volume of 200 ml. 0.05M Tris-HCl buffer

x	y	pH	x	y	pH
50.0	5.0	9.0	50.0	26.8	8.0
50.0	8.1	8.8	50.0	32.8	7.8
50.0	12.2	8.6	50.0	38.4	7.6
50.0	16.5	8.4	50.0	41.4	7.4
50.0	21.9	8.2	50.0	44.2	7.2

Adjust pH with pH meter by adding required component if necessary

4. Make stock solutions for Citrate-phosphate Buffer.

A-0.2 M dibasic sodium phosphate

B-0.1M citric acid

X ml of A + y ml of B, dilute to a total volume of 200 ml

x	y	pH	x	y	pH
20.55	79.45	3.0	63.15	36.85	6.0
38.55	61.45	4.0	82.35	17.65	7.0
51.50	48.50	5.0	97.25	2.75	8.0

Adjust pH with pH meter by adding required component if necessary

Observation:

Note:

Components	pH range
HCl, Sodium citrate	1 - 5
Citric acid, Sodium citrate	2.5 - 5.6
Acetic acid, Sodium acetate	3.7 - 5.6
K2HPO4, KH2PO4	5.8 - 8
Na2HPO4, NaH2PO4	6 - 7.5
Borax, Sodium hydroxide	9.2 - 11
Tris Buffer	7.2-9.0

Experiment no. 4

Preparation of various sub-cellular fractions of liver cells (rat, chicken…) by

Differential Centrifugation method

Objectives:

1. To prepare various sub-cellular fractions of liver cells (rat, chicken…) by Differential centrifugation method

Principle:

The process of differential centrifugation is based on the fact that organelles have differences in size, shape and density. As a result, the effect of gravity on each is different. We can use this principle to separate an organelle from a homogenous solution of particles by artificially controlling the gravity of a solution. This is done by putting the solution in a variable speed centrifuge and rotating them at a high rate of speed. This creates a force that can be much greater than the force of gravity, and particles that would normally stay in solution will fall out and form a pellet at the bottom of the tube.

Differential centrifugation schemes involve stepwise increases in the speed of centrifugation. At each step, more dense particles are separated from less dense particles, and the successive speed of centrifugation is increased until the target particle is pelleted out. The final supernatant is removed, the pellet is resuspended and further study or purification can be done on it. The fractionation of rat liver is an example of how this process works.

Materials and Reagents:

1. Centrifuge machine

2. Centrifuge tubes

3. Homogenizer

4. Homogenization media: 5mM Tris-HCl buffer(pH 7.4) containing 0.25M sucrose

5. Liver cells

6. Cheese cloth

Procedure:

1. Weigh 1 g of liver tissue (Chicken liver) and cut into small pieces.

2. Transfer the liver slices alongwith homogenization media into chilled homogenizer. Operate the homogenizer and push the glass tube (handle) up and down to ensure the breakage of the cells.

3. Filter the homogenate through 3-4 layers of cheese cloth.

4. Pour the homogenate into a centrifuge tube and centrifuge the supernatant at 1000 X g for 10 min to sediment the heaviest material (P1-pellet- 1).

5. Remove the supernatant carefully and again centrifuge it at 3000 X g for 10 min to obtain second heaviest material (P2 pellet-2). Recover the pellet by withdrawing the supernatant with a syringe or micropipette.

6. Subject the supernatant from the preceding step to centrifuge at 10000 X g for 30 min.Gently recover the pellet by withdrawing the supernatant and labeled it P3 (pellet-3)

7. Finally centrifuge the remaining supernatant from the above step at 100000 X g for 40 min to obtain pellet P4. Decant the supernatant into a chilled beaker and store it.

8. Store all pellets separately at chilled condition and observe for the contents.

Experiment no. 5

Fractionation of serum protein by salt (Sodium chloride and Ammonium sulphate=separation of albumin and globulin)

Objectives:

1. To separate albumin and globulin of serum protein by Ammonium sulphate fractionation

2. To separate albumin and globulin of serum protein by Sodium chloride

Principle:

There are hydrophobic amino acids and hydrophilic amino acids in protein molecules. After protein folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules. If enough of the protein surface is hydrophilic, the protein can be dissolved in water. When the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein. As a result of the increased demand for solvent molecules, the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other. This process is known as salting out. As different proteins have different compositions of amino acids, different protein molecules precipitate at different concentrations of salt solution. Unwanted proteins can be removed from a protein solution mixture by salting out as long as the solubility of the protein in various concentrations of salt solution is known. After removing the precipitate by filtration or centrifugation, the desired protein can be precipitated by altering the salt concentration to the level at which the desired protein becomes insoluble.

Certain ions can increase the solubility of a protein when the concentration of the ions increases, instead of decreasing the solubility of the protein. Also some ions can denature certain proteins so if assays on the function of proteins are intended then either a different ion or an alternative purification method should be used. In attempting to remove a product from water, NaCl is often used to increase the ionic strength of water, thereby increasing its polarity, and then the product is moved into the organic layer where it can be extracted.

Materials and Reagents:

1. Centrifuge machine
2. Centrifuge tubes
3. Dialysis bags
4. Ammonium sulphate / Sodium chloride
5. Acetic acid / Barium chloride / Silver nitrate
6. Blood sample

Procedure:

Fractionation by Ammonium sulphate:

1. Take 2 ml of fresh blood serum in a test tube.

2. Add 2 ml of saturated ammonium sulphate in the serum sample and stir well.

3. Transfer the serum sample into a centrifuge tube as soon as precipitate appears and centrifuge at 4000 rpm for 10 min to obtain the pellet (globulin fraction).

4. Store the pellet at cool condition with label.

5. Again take the supernatant from the previous step and add the ammonium sulphate crystal slowly till precipitate observe.

6. Centrifuge again at 8000 rpm for 10 min.

7. Collect the pellet 2 (Albumin fraction) and store at cool condition.

8. Dilute each fraction in appropriate buffer and fill in dialysis bags separately.

9. Then, perform dialysis to remove the salts in the precipitates.

Fractionation by Sodium chloride:

1. Take 2 ml of fresh blood serum in a test tube.

2. Add sodium crystals in the serum sample and stir well until saturation.

3. Transfer the serum sample into a centrifuge tube as soon as precipitate appears and centrifuge at 4000 rpm for 10 min to obtain the pellet (globulin fraction).

4. Store the pellet at cool condition with label.

5. Again take the supernatant from the previous step and add acetic acid slowly till precipitate observe.

6. Centrifuge again at 8000 rpm for 10 min.

7. Collect the pellet 2 (Albumin fraction) and store at cool condition.

8. Dilute each fraction in appropriate buffer and fill in dialysis bags separately.

9. Then, perform dialysis to remove the salts in the precipitates.

Observation:

Serum is rich in proteins. About 6-8 % of the serum is protein. Among the proteins, the major two proteins in serum are albumin and globulin. Albumin constitutes 35-60 g/L of serum while globulin constitutes about 25-35 g/L of serum. Hence the ratio of the two proteins in serum should be; Albumin: Globulin=2: 1 to 2.5: 1.

Table-1: Ammonium Sulphate Concentration table

Final required concentration of ammonium sulphate (% saturation)
%	10	15	20	25	30	33	35	40	45	50	55	60	65	70	75	80	85	90	95	100
Grams solid ammonium sulphate to be added to 1 L of solution
0	56	84	114	144	176	196	209	243	277	313	351	390	430	472	516	561	610	662	713	767
10		28	57	86	118	137	190	183	216	251	288	326	365	406	449	494	540	592	640	694
15			28	57	88	107	120	153	185	220	256	294	333	373	415	459	506	556	605	657
20				29	59	78	91	123	155	189	225	262	300	340	382	424	471	520	569	619
25					30	49	61	93	125	158	193	230	267	307	348	390	436	485	533	583
30						19	30	62	94	127	162	198	235	273	314	356	401	449	496	546
33							12	43	74	107	142	177	214	252	292	333	378	426	472	522
35								31	63	94	129	164	200	238	278	319	364	411	457	506
40									31	63	97	132	168	205	245	285	328	375	420	469
45										32	65	99	134	171	210	250	293	339	383	431
50											33	66	101	137	176	214	256	302	345	392
55												30	67	103	141	179	220	264	307	353
60													34	69	105	143	183	227	269	314
65														34	70	107	147	190	232	275
70															35	72	110	153	194	237
75																36	74	115	155	198
80																	38	77	117	157
85																		39	77	118
90																			38	77
95																				39

Experiment no. 6

Precipitation and separation of protein of interest by TCA (Trichloroacetic acid)

method from biological sample

Objectives:

1. To precipitate the protein of interest by TCA (Trichloroacetic acid) method

Principle:

The solubility of proteins is determined by four variables: pH, ionic strength, temperature, and protein concentration. Numerous different techniques have been developed for protein precipitation by modifying one or more of these parameters. TCA leads to a strong decrease in pH, resulting in denaturation and consequently precipitation of the protein. Three chloro groups in the molecule also play an important role in protein precipitation

Materials and Reagents:

1. Centrifuge machine
2. Centrifuge tubes
3. Trichloroacetic acid
4. Acetone
5. serum
6. 0.1M phosphate buffer

Procedure:

1. Take 2 ml in a clean test tube and add volumes of ice cold acetone containing 10% w/v TCA.

2. Mix immediately by gentle vortexing and incubate at -20^oC for 90 minute

3. Centrifuge at 8,000 rpm, 4^oC for 10min and collect the pellet (Globulin)

4. Remove the supernatant. Again add equal volume of ice cold acetone to pellet to wash the precipitate.

5. Incubate and centrifuge as above.

6. Remove the acetone containing supernatant.

7. Add equal volume of ice cold acetone to the 10% TCA/acetone-containing supernatant to completely precipitate the protein in the supernatant.

8. The precipitate (Albumin) was dissolved in minimum volume of 0.1 M phosphate buffer, pH 7.00.

Experiment no. 7

Separation and identification of amino acids in a given mixture by ascending paper chromatography

Objectives:

1. To separate amino acids in a given mixture by ascending paper chromatography.

2. To identify the amino acids by comparing their R_f values with standard amino acids.

Principle:

Amino acids in a given mixture or sample aliquot are separated on the basis of differences in their solubilities hence differential partitioning coefficients in a binary solvent system. The amino acids with higher solubilities in stationary phase move slowly as compared to those with higher solubilities in the mobile phase. The separated amino acids are detected by spraying the air dried chromatogram with ninhydrin reagent. All amino acids give purple or bluish purple colour on reaction with ninhydrin except praline and hydroxyproline which give a yellow coloured product. The reactions leading to the formation of purple complexes are given below:

Materials and Reagents:

1. Whatman No. 1 filter paper sheet
2. Micropipette / microsyringe
3. Hair drier / Sprayer
4. Oven set at 105˚C
5. Chromatographic chamber saturated with water vapors
6. Developing solvent: butanol, acetic acid and water in the ratio of 4:1:5
7. Ninhydrin spray reagent: Prepare fresh by dissolving 0.2g ninhydrin in 100 ml acetone
8. Standard amino acids: Prepare solutions of authentic samples of amino acids such as methionine, tryptophan, alanine, glycine, threonine etc.(1 mg/ml of 10% iso-propanol)
9. A sample containing mixture of unknown amino acids

Procedure:

1. Take Whatman No. 1 filter paper and lay it on a rough filter paper. Throughout the experiment care should be taken not to handle the filter paper with naked hands and for this purpose either gloves should be used or it should be handled with the help of folded piece of rough filter paper.
2. Fold the Whatman No.1 filter paper about 2.0-2.5 cm from one edge. Reverse the paper and again fold it 2 cm further down from the first fold.
3. Draw a line across the filter paper with a lead pencil at a distance of about 2 cm from the second fold. Put circular marks along this line at a distance of 2.5 cm from each other.
4. With the help of a micropipette or microsyringe apply 20 μl of solution of each standard amino acid on a separate mark. Also apply spot of the sample or mixture to be analyzed, preferably on the mark at the center of this base line. The size of the spot should be as small as possible so that the developed spots are compact and do not overlap. If necessary, the wet sample spot should be dried with hair drier before applying additional aliquot.
5. Hang the filter paper in a chromatographic chamber which has preciously been saturated with aquerous phase of the solvent system. This is done by keeping Petri plates containing the aqueous phase at bottom of the chamber. The paper is hung from the trough/tray and a glass rod is kept to hold it in place. Care should be taken to ensure that the base line should not get submerged when the mobile phase added to the trough otherwise the spotted material would get dissolved in the solvent.
6. Close the chamber firmly so that it is airtight. Allow sufficient time for cellulose fibers of the paper to get fully hydrated.
7. Pour the mobile phase through the holes provided on the lid of the chamber into the trough. Replace the rubber bungs in the hole and allow the mobile phase to run down the paper till solvent front reaches about 5 cm from the opposite edge.
8. Remove the paper and mark the solvent front with lead pencil and let it dry at room temperature.
9. Spray the filter paper (chromatogram) with ninhydrin reagent and after drying it at room temperature, transfer it to an oven at 105˚C for 5-10 min.
10. Blue or purple colored spots would appear on the paper. Mark the boundary of each spot with lead pencil.
11. Measure the distance between the center of the spots and also the distance of the solvent front from the base line.
12. Calculate the R_f value of standard amino acids as well as those in the given mixture or sample as follows:
13. Identify the amino acids in the mixture or sample by comparing their R_f values with those of the reference standards.

Note: It is advisable to carry out chromatography in three different solvent systems before the identity of amino acid in the mixture or sample can be established with any degree of certainty.

Experiment no. 8

Separation and identification of amino acids in a given mixture by two dimensional

paper Chromatography

Objectives:

1. To separate amino acids in a given mixture by two dimensional paper chromatography.

2. To identify the amino acids by comparing their R_f values with standard amino acids.

Principle:

Amino acids have very close Rf values in a particular solvent system may appear as a single or overlapping spots in a single dimensional chromatography and may be mistaken as one component. They can be separated into individual components by developing the chromatogram again in a direction perpendicular to the first run in a second solvent system in which they have different R_f values. The main limitation of this method is that only one spot either filter paper sheet necessitating running of a large number of chromatogram for the standard amino acids.

Materials and Reagents:

1. Whatman No. 1 filter paper sheet
2. Micropipette / microsyringe
3. Hair drier / Sprayer
4. Oven set at 105˚C
5. Chromatographic chamber saturated with water vapours
6. Solvent system first: butanol: acetic acid: water= 4:1:5 v/v
7. Solvent system second: phenol: water=80:20 w/v (Add 125 ml of water to 500g of phenol and add few drops of ammonia to the mixture)
8. Ninhydrin spray reagent: Prepare fresh by dissolving 0.2g ninhydrin in 100 ml acetone
9. Standard amino acids: Prepare solutions of authentic samples of amino acids such as methionine, tryptophan, alanine, glycine, threonine etc.(1 mg/ml of 10% iso-propanol)
10. A sample containing mixture of unknown amino acids

Procedure:

1. Take Whatman No. 1 filter paper and lay it on a rough filter paper. Throughout the experiment care should be taken not to handle the filter paper with naked hands and for this purpose either gloves should be used or it should be handled with the help of folded piece of rough filter paper.
2. Draw a base line 5 cm from one edge of the paper. Reverse the paper and again draw another line perpendicular to the first line again 5 cm from one adjacent edge of the paper.
3. With the help of a micropipette or microsyringe apply 60 μl of solution at the point of intersection.
4. Repeat the same procedure for a mixture of standard amino acids in a separate chromatographic sheet for each mixture.
5. Hang the chromatographic sheets in a chromatographic chamber which has preciously been saturated with aqueous phase of the solvent system first. This is done by keeping Petri plates containing the aqueous phase at bottom of the chamber. The paper is hung from the trough/tray and a glass rod is kept to hold it in place.
6. After allowing an equilibrium period of half an hour, pour the solvent system first in to the trough of the chamber and let it run till it is about 5cm from the opposite end of the paper.
7. Take the paper out, air dry it and turn it at 90˚ and now develop the paper in the second chromatographic chamber using solvent system second.
8. Remove it when the solvent has traveled upto about 5 cm from the opposite end.
9. Spray the filter paper (chromatogram) with ninhydrin reagent and after drying it at room temperature, transfer it to an oven at 105˚C for 5-10 min.

Observation:

Calculate the R_f value of standard amino acids as well as those in the given mixture in both the solvents and identify the amino acids in the mixture or sample by comparing their R_f values with those of the reference standards.

Experiment no. 9

Separation and identification of sugars (sugar juices) by adsorption Thin layer chromatography (TLC)

Objectives:

1. To separate sugars in a given mixture by adsorption Thin layer chromatography.

Principle:

Sugars get separated on the basis of differential adsorption onto silica gel. The sugars which have higher affinity for stationary phase are adsorbed more strongly and hence they migrate slowly when mobile phase moves over them. On the other hand, those having lower affinity for stationary phase are weakly adsorbed and are more easily carried by the mobile phase. The separated sugars are then located as colored zones by spraying TLC plates with aniline diphenylamine reagent.

Materials and Reagents:

1. TLC chromatographic tank
2. Glass plates (20 х 20 cm)
3. Spreader / Hair drier / Sprayer or atomizer
4. Micropipettes / micro-syringes
5. Oven maintained at 105˚C
6. Solvent System: Prepare a mixture of ethyl acetate: iso-propanol: water: pyridine (26:14:7:2,v/v)
7. Standard sugar solutions: Prepare 1% solution of standard sugars such as glucose, ribose, lactose etc. in 10% iso-propanol (v/v). For mixture in which the sugars have to be identified, mix the sugar solutions in equal proportion
8. Aniline diphenylamine reagent: Mix 5 volumes of 1% aniline and 5 volumes of 1% diphenylamine in acetone with 1 volume of 85% phosphoric acid

Procedure:

1. Place thoroughly cleaned and dried glass plates (20x20 cm) on a flat plastic tray side by side with no gap between the two adjacent plates.
2. Prepare slurry of the stationary phase (Silica Gel G) free of clumps in water or in an appropriate buffer.
3. Spread a uniform layer of 250 µm thickness with the help of a spreader or an applicator by moving it from one end of the ray of the tray to its other end.
4. Activate the plates by keeping them at 105˚C for 30 min. Allow the plates to cool in a desiccator before use.
5. Gently put the marks in a straight line with the help of a pin at a distance of about 2 cm from one edge of the plate. The adjacent marks should be taken that silica does not get scratched of while putting these marks.
6. Carefully apply the solution of individual standard sugars and the mixture or alcoholic extract of the sample on the separate marked spots.
7. Gently put marks or draw a line 1 cm from the opposite edge.
8. Place the plate in chromatographic tank which has already been equilibrated with the solvent taking care that base line on which samples have been applied does not dip into the solvent.
9. Close the chromatographic tank with air tight lid and allow the solvent to ascend along the plate by capillary action till the solvent reaches the marked line on the upper side of the plate. This may take about 90 min.
10. Remove the plate from chromatographic tank and let it dry at room temperature.
11. For determining the location of sugars on the TLC plates, spray it with freshly prepared aniline-diphenylamine reagent ensuring that silica gel is not removed or brown off while spraying.
12. Place the plates in hot air oven at 100˚C for 10 min. Appearance of bluish spots on the white background indicates presence of sugars at that region of the plate.
13. Measure the distance from the base line to the center of the colored spot and calculate the R_f value of each sugar as described in EXPERIMENT 9.6.1.
14. Identify the sugars in the given mixture or sample by comparing their R_f values with those of sugar standards.

Precautions:

A number of precautions should be observed while performing TLC. Some of these are:

1. Thoroughly cleaned glass plates free of any greasy spots or finger marks should be used.

2. Thickness of the layer should be uniform throughout the length of the plate.

3. The slurry of the chromatographic media should be free of any clumps. This can be ensured by vigorously shaking it in an Erlenmeyer flask or by gently preparing the slurry in pestle and mortar to ensure uniform mixing.
4. The TLC plates should be activated at recommended temperature and duration. Poor resolution of components occurs on over or under activated plates.
5. The layer of chromatographic media should not get scraped off at the time of putting marks of application of samples.
6. Size of the applied spot should be as small as possible. If large volume of the sample has to be spotted, then it should be done in small aliquots with an intermittent drying. Overloading of the sample should be avoided.

The chromatographic tank should be airtight and chromatography should be performed under temperature controlled conditions.

Experiment no. 10

Extraction and identification of lipids from given biological samples (egg, cooking

oils…..) by Thin layer chromatography

Objectives:

1. To extract lipids from biological samples

2. To identify the lipids by Thin layer chromatography

Principle:

In the biological materials, lipids are found as lipoprotein complexes and these have to be extracted. Lipids, being soluble in non-polar organic solvents and proteins being soluble in polar aqueous solvents, the efficient lip[id extraction can be achieved only when an aqueous solvent like ethanol or methanol is included in th non-polar organic solvents like chloroform and diethyl ether. This would help in breaking the lipoprotein complexes. Extracted lipid components can be separated on TLC based on their differential mobility along the porous stationary phase such as silica gel and these can be located by spraying the plates with either 2’, 7’ dichlorofluorescein or 50% sulphuric acid or iodine vapor.

Materials and Reagents:

1. TLC chromatographic tank
2. Glass plates (20 х 20 cm)
3. Micropipettes / microsyringes / Spreader / Hair drier / Sprayer or atomizer
4. Oven maintained at 105˚C
5. Solvent System: petroleum ether (b.p-60-70˚C) or hexane: diethyl ether: glacial acetic acid(80:120:1 v/v)
6. Lipid standards: varied lipids such as cholesterol acetate, vitamin A, palmitate, triacyl glycerol, vegetative oils, egg, animal oils.
7. Staining reagent: 2’, 7’ dichlorofluorescein or 50% sulphuric acid or iodine crystals.

Procedure:

1. Extraction of lipids from sample: Grind 1 g of the tissue in the extraction solvents (either ethyl ether: ethanol=3:1 or chloroform: methanol=2:1) in mortal and pestle. Transfer the homogenate to a separating funnel. Shake the contents vigorously and allow it to stand till the two phases have completely separated. Drain out the lower organic layer which contains the lipids. Evaporate the solvent under vaccum and keep the concentrated lipid extract protected from light under N atmosphere.
2. Extraction from egg yolk: Make a small hole on the polar region of egg and invert in into a small beaker to separate white part (sac) till yellow part fall down. Then, mix and dissolve yolk in 5 ml of chloroform. Centrifuge it at 4000rpm for 10 min and evaporate on boiling water bath. Again redissolve in required amount of chloroform.
3. Prepare the TLC plate using silica gel as described in previous experiment-9 and activate the plates by keeping them at 105˚C for 30 min. Allow the plates to cool in a desiccator before use.
4. Carefully apply the solution of individual lipid samples on the separate marked spots.
5. Develop the plates in the solvent system described above till the solvent traveled upto 1 cm from the opposite side of the plate.
6. Remove the plate from chromatographic tank and let it dry at room temperature.
7. For determining the location of lipids on the TLC plates, spray it with freshly prepared 2’, 7’ dichlorofluorescein or 50% sulphuric acid. Or place the tank in en empty tank and add iodine crystal into the tank, then shield the tank airtightly by glass plate.
8. Calculate the R_f values of lipids standards and identify the lipids by comparing their with those of standards.

Experiment no. 11

Purification of bovine serum albumin from buffalo serum by size exclusion

chromatography (gel filtration)

Objectives:

1. To purify bovine serum albumin from buffalo serum by size exclusion chromatography (Gel Filtration).

Principle:

The chromatographic media used in this technique are porous, polymeric organic compounds with molecular sieving properties. These are cross linked polymers which swell considerably in water forming a gel of a three dimensional network of pores. The size of pore is determined by degree of cross-linking of polymeric chains. Differential solutes in a mixture get separated on the basis of their molecular size and shape during their passage through a column packed with the swollen particles. The terms “exclusion chromatography”, “gel filtration” and “molecular sieve” chromatography are used for this separation process. The large molecules in the sample are unable to penetrate through the pores into the gel and thus remain excluded from entering the beads. Therefore they travel through the interstitial spaces with high acceleration. However the small molecules enter into the gel beads and get distributed between the mobile phase inside as well as outside the gel particles. These thus follow a longer path than the larger molecules and hence their movement down is retarded. Consequently different molecules getting separated in the sample get separated from each other with larger molecule getting eluted first followed by smaller molecules.

Material and Reagents:

1. Glass column (2.5 X 25 cm)

2. Spectrophotometer

3. Sephadex G-100

4. Buffalo Serum

5. Sodium phosphate

6. 0.1 M Tris HCl buffer (pH 7.0)

7. SDS-PAGE (will be described in later)

Procedure:

1. Suspend 5 g of sephadex G-100 (coarse) in 0.1 M Tris-HCl buffer (pH 7.0) and swell it by keeping it for 3-4 h at room temperature with intermittent stirring.
2. Decant the excess of buffer alongwith any suspended fine particles to obtain slurry of reasonable thickness.
3. Fix the column upright on a burette stand with the help of clamps.
4. Keep the outlet of the column closed, place a plug of glass wool at he base of the column and pour a small volume of the buffer or water into the column.
5. Pour the slurry gradually into it along the inner surface of its wall and, if necessary with gentle tapping to expel any air bubbles.
6. Allow the chromatographic media to settle down evenly and then open the outlet to drain excess liquid from the column.
7. Place a filter paper disc or a nylon gauze on the surface of the packed bed to prevent disturbance of the upper layer while loading the sample or feeding the eluent into the column.
8. Prepare a mixture of 10mg of bovine serum and 40 mg of sodium phosphate in 2 ml Tris-HCl buffer (pH 7.0).
9. Apply it to chromatographic column by any one of the two methods:

i. The mobile phase at the top of the packing is drained out till the bed surface gets exposed. Close the outlet and gently apply the sample uniformly over the bed surface with pipette and the loaded sample is then allowed to just enter into the column by opening the outlet. A small amount of mobile phase is added to wash the traces of the sample into the column.

ii. In the second method, sucrose or glycerol, upto the concentration of 1% is added in the sample to increase its density. This sample is applied just above the surface of bed directly through the layer of the buffer in the column bed. Since the sample has higher density, it automatically settles on the surface of the gel. Then open the outlet to facilitate entry of the sample into the column. When using this procedure, it is advisable to ensure that addition of glycerol or sucrose does not interfere with the separation and subsequent analysis of the separated compounds.

10. Add sufficient amount of buffer on top of the column and connect it to the buffer reservoir.
11. Collect the fraction of 2 ml either manually or using an automatic fraction collector. Determine the protein content either by monitoring absorbance at 280 nm or any chemical detection method.
12. Plot a graph of concentration of protein and fraction number or elution volume.
13. Find out the molecular weight of each peak fraction by SDS-PAGE.

Experiment no. 12

Determination of molecular weight of a given protein by SDS-PAGE (Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis)

Objective:

1. To determine the molecular weight of a given protein by SDS-PAGE

Principle:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has proven to be among the most useful tools yet developed in the area of molecular biology. The discontinuous buffer system, first described by Laemmli. Acrylamide mixed with bisacrylamide forms a cross-linked polymer network when the polymerizing agent, ammonium persulfate, is added (Figure 1). The ammonium persulfate produces free radicals faster in the presence of TEMED (N, N, N, N’-tetramethylenediamine). The size of the pores created in the gel is inversely related to the amount of acrylamide used. Gels with a low percentage of acrylamide are typically used to resolve large proteins and high percentage gels are used to resolve small proteins.

Figure 1. Polymerization and cross-linking of acrylamide.

The electrophoretic mobility of a protein is determined mostly by its net charge per unit mass at the given pH, but is inversely proportional to its frictional coefficient in the gel, determined by the proteins size and shape. In the denaturing, reductive variant of PAGE, SDS-PAGE all differences between the proteins in charge per unit mass has been eliminated by the SDS (sodium dodecylsulphate) and the proteins migrate solely according to subunit size. The charged SDS molecules bind all along the polypetide chain, giving the chain equal charge per unit length. Thus, the denatured polypeptide chains are separated electrophoretically only according to their subunit length

Materials and Reagents:

1. SDS-PAGE gel apparatus.
2. Power pack.
3. Blotting apparatus.
4. Separating gel
5. Stacking gel
6. Running buffer
7. Sample buffer
8. Staining solution
9. Destaining solution

1. Separating Gel (pH 8.8)

	7%	10%	12%	15%
Distilled water	5.1 ml	4.1 ml	3.4 ml	2.4 ml
1.5 M Tris-HCl, pH 8.8	2.5 ml	2.5 ml	2.5 ml	2.5 ml
20% (w/v) SDS	0.05 ml	0.05 ml	0.05 ml	0.05 ml
Acrylamide/Bis-acrylamide (30%/0.8% w/v)	2.3 ml	3.3 ml	4.0 ml	5.0 ml
10% (w/v) ammonium persulfate	0.05 ml	0.05 ml	0.05 ml	0.05 ml
TEMED	0.005 ml	0.005 ml	0.005 ml	0.005 ml
Total monomer	10.005 ml	10.005 ml	10.005 ml	10.005 ml

2. The Stacking Gel (pH 6.8)

Ingredients Amount

Distilled water 3.075 ml

0.5 M Tris-HCl, pH 6.8 1.25 ml

0% (w/v) SDS 0.025 ml

Acrylamide/Bis-acrylamide (30%/0.8% w/v) 0.67 ml

10% (w/v) ammonium per sulfate 0.025 ml

TEMED 0.005 ml

Total Stack monomer 5.05 ml

For best results:

1. Ammonium persulfate solution should be freshly made.

2. Degas solutions should be degassed before adding TEMED for 15 min at room temperature.

3. Running the gel (5X, pH 8.3)

Ingredients Amount

Tris Base 15 g

Glycine 72 g

SDS 5 g

Distilled water 1000 ml

Dilute to 1X before use and store at room temperature until use.

4. Sample buffer

Ingredients Amount

Distilled water 4.0 ml

Tris-HCl (0.5 M) 1.0 ml

Glycerol 0.8 ml

10% SDS 1.6 ml

β-mercaptoethanol 0.4 ml

Bromophenol blue (0.05%) 0.2 ml

5. Staining Solution

Ingredients Amount

Coomasie Brilliant Blue R-250 0.5 g

Methanol 250 ml

Acetic acid 50 ml

Distilled water 200 ml

6. Destaining Solution

The destaining solution was prepared similarly but without dye.

Procedure:

Sample Solubilization

1. Boil samples in sample solubilization buffer for 5-10 min. Solubilize sample at 1 mg/mL and run 5–10 μg/lane.

Gel Preparation/Electrophoresis

2. Assemble the gel apparatus. Make two marks on the front plate to identify top of separating gel and top of spacer gel. Assuming a well depth of 12 mm, the top of the separating gel should be 3.5 cm down from the top of the back plate, and the spacer gel should be 2 cm down from the top of the back plate, leaving a stacking gel of 8 mm.
3. Combine the reagents to make the separating gel, mix gently, and pipette the solution between the plates to lowest mark on the plate. Overlay the gel solution with 2 mL of dH₂O by gently running the dH₂O down the center of the inside of the front plate. Allow the gel to polymerize for about 20 min. When polymerized, the water–gel interface will be obvious.
4. Pour off the water, and dry between the plates with filter paper. Do not touch the surface of the separating gel with the paper. Combine the reagents to make the spacer gel, mix gently, and pipette the solution between the plates to second mark on the plate. Overlay the solution with 2 mL of dH2O by gently running the dH₂O down the center of the inside of the front plate. Allow the gel to polymerize for about 20 min. When polymerized, the water–gel interface will be obvious.
5. Pour off the water, and dry between the plates with filter paper. Do not touch the surface of spacer gel with the paper. Combine the reagents to make the stacking gel and mix gently. Place the well-forming comb between the plates, leaving one end slightly higher than the other. Slowly add the stacking gel solution at the raised end (this allows air bubbles to be pushed up and out from under the comb teeth). When the solution reaches the top of the back plate, gently push the comb all the way down. Check to be sure that no air pockets are trapped beneath the comb. Allow the gel to polymerize for about 20 min.
6. When the stacking gel has polymerized, carefully remove the comb. Straighten any wells that might be crooked with a straightened metal paper clip. Remove the acrylamide at each edge to the depth of the wells. This helps prevent “smiling” of the samples at the edge of the gel. Seal the edges of the gel with 2% agarose.
7. Add freshly diluted cathode running buffer to the top chamber of the gel apparatus until it is 5–10 mm above the top of the gel. Squirt running buffer into each well with a Pasteur pipet to flush out any unpolymerized acrylamide. Check the lower chamber to ensure that no cathode running buffer is leaking from the top chamber, and then fill the bottom chamber with anode buffer. Remove any air bubbles from the under edge of the gel with a bent tip Pasteur pipette. The gel is now ready for sample loading.
8. After loading the samples and the molecular-mass markers, connect leads from the power pack to the gel apparatus (the negative lead goes on the top, and the positive lead goes on the bottom). Gels can be run on constant current, constant voltage, or constant power settings. When using the constant current setting, run the gel at 50 mA. The voltage will be between 50 and 100 V at the beginning, and will slowly increase during the run. For a constant voltage setting, begin the electrophoresis at 50 mA. As the run progresses, the amperage will decrease, so adjust the amperage to 50 mA several times during the run or
9. The electrophoresis will be very slow. If running on constant power, set between 5 and 7 W. Voltage and current will vary to maintain the wattage setting. Each system varies, so empirical information should be used to modify the electrophoresis conditions so that electrophoresis is completed in about 4 h.
10. When the dye front reaches the bottom of the gel, turn off the power, disassemble the gel apparatus, and place the gel in 200–300 mL of fixer/destainer. Gently shake for 16 h. Pour off spent fixer/destainer, and add CBB. Gently shake for 30 min. destain the gel in several changes of fixer/destainer until the background is almost clear. Then place the gel in dH₂O, and gently mix until the background is completely clear. The peptide bands will become a deep purple-blue. The gel can now be photographed or dried. To store the gel wet, soak the gel in 7% glacial acetic acid for 1 h, and seal in a plastic bag.

Experiment no. 13

Native Disc gel electrophoresis of proteins

Objective:

1. To perform the nondenaturing gel electrophoresis of protein

Principle:

Nondenaturing system is used to separate intact proteins, especially oligomeric proteins, by a nondestructive means for later assessment of biological activity. On nondenaturing system, protein separation depends on a combination of differences in molecular size and shape as well as charge. Separation by size is accomplished by varying the pore size of the acrylamide polymer as a function of both the concentration of the acrylamide (range about 3-30%, w/v) and the amount of cross-linker used. In general, the higher the acrylamide concentration, the smaller the protein that remains in gel: this can be counteracted by decreasing the amount of cross-linker used, which in turn increases the degree of gel swelling during standard staining and washing procedures. Separation by charge in nondenaturing gel systems is permitted because the protein separation can be performed at any pH between 3 and 11 to allow for maximal charge differences neighboring protein species. These include the molecular weight of the stability in the range of pH 4 to 9. Once these parameters are known optimal resolution can be obtained by varying certain components within a single gel system. For example the initial choice of the gel system for separation of acidic protein of molecular weight 20,000 might be a system operating at an alkaline pH i.e.9 with an acrylamide concentration of 12-15 %.

Materials and Reagents:

1. Same as in SDS-PAGE

1. The Stacking Gel (pH 6.8)

Ingredients Amount

Distilled water 3.075 ml

0.5 M Tris-HCl, pH 6.8 1.25 ml

Acrylamide/Bis-acrylamide (30%/0.8% w/v) 0.67 ml

Riboflavin (0.004%) 0.0625 ml

TEMED 0.005 ml

Total Stack monomer 5.05 ml

2. Separating Gel (pH 8.8)

	7%	10%	12%	15%
Distilled water	5.1 ml	4.1 ml	3.4 ml	2.4 ml
1.5 M Tris-HCl, pH 8.8	2.5 ml	2.5 ml	2.5 ml	2.5 ml
Acrylamide/Bis-acrylamide (30%/0.8% w/v)	2.3 ml	3.3 ml	4.0 ml	5.0 ml
10% (w/v) ammonium persulfate	0.05 ml	0.05 ml	0.05 ml	0.05 ml
TEMED	0.005 ml	0.005 ml	0.005 ml	0.005 ml
Total monomer	10.005 ml	10.005 ml	10.005 ml	10.005 ml

Remainings are same as for SDS-PAGE

Procedure: Same as for SDS-PAGE.

Experiment no. 14

Determination of molecular weight of DNA (genomic and plasmid) by Agarose gel

electrophoresis

Objectives:

1. To determine molecular weight of DNA by agarose gel electrophoresis

Principle:

DNA electrophoresis is an analytical technique used to separate DNA fragments by size. An electric field forces the fragments to migrate through a gel. DNA molecules normally migrate from negative to positive potential due to the net negative charge of the phosphate backbone of the DNA chain. At the scale of the length of DNA molecules, the gel looks much like a random, intricate network. Longer molecules migrate more slowly because they are more easily 'trapped' in the network. Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.

After the separation is completed, the fractions of DNA fragments of different length are often visualized using a fluorescent dye specific for DNA, such as ethidium bromide. The gel shows bands corresponding to different DNA molecules populations with different molecular weight. Fragment size is usually reported in "nucleotides", "base pairs" or "kb" (for 1000's of base pairs) depending upon whether single- or double-stranded DNA has been separated. Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length.

Materials and Reagents:

1. 1X TAE buffer at pH 8.0 (50X, 24.2gm Tris, 5.71ml GAA, 11.1ml of 0.5M EDTA, 100ml DW) Autoclave before use – 1.5L
2. Agarose gel in 1X TAE – 65ml
3. Loading dye (6X, 6.0ml of 50% Glycerol, 1.0ml of 2%BPB, 3ml sterile DW) – 0.5ml
4. Ethidiun Bromide (EtBr)*: 10mg/ml stock
5. λ/HindIII Marker (23.13Kb, 9.42Kb, 6.56Kb, 2.32Kb, 2.07Kb, 0.56Kb and 0.13Kb)
6. Plasmid DNA (pUC18, pBR322)

Procedure:

1. Prepare 0.8% agarose gel in TAE buffer.

2. Dissolve agarose completely in micro-oven and cool to 60⁰C.

3. CAREFULLY, add EtBr into the gel solution to final concentration of 0.5µg/ml.

4. Pour the molten gel into gel-mould. Immediately position a comb in the mould.

5. Let the gel cool for 30 minutes.

6. Pour TAE buffer into the gel buffer reservoirs.

7. Prepare sample taking 20µl of DNA sample and mix with 4µl blue juice (6X).

8. Carefully remove the comb.

9. Load the DNA (15 - 20 µl) per well, flanking wells with similarly processed DNA size standard.

10. Put the lid on the gel apparatus, attach the electrodes and adjust voltage to 100 volts.

11. Allow the gel to run until line of blue juice is visible near the end of the gel.

12. Turn off the current and visualize the gel in UV - Tran illuminator.

13. Interpret the results.

Note: the amount of the sample that can be loaded in a well depends on the thickness of the gel as well as dimensions and placing of the comb.: EtBr is carcinogen, so, handle with gloves

Experiment no. 15

Determination of l_max of different colored and noncolored compounds by spectrophotometer

Objectives:

1. To determine l_max of different colored and noncolored compounds by spectrophotometer

Principle:

For a given substance at a specified wavelength λ, the absorptivity ε_λ is a constant characteristic of the absorbing sample and is independent of both the concentration c of the solution and the thickness d of the absorbing layer. Absorptivity and, the absorption itself depend strongly on the wavelength for nearly all compounds. So, the wavelength must be specified at which the measurement of the absorbance versus concentration is made. The way in which absorbance depends on wavelength, A= f(c), defines the spectrum of the substance being studied and the wave length at which maximum absorbance in the electromagnetic spectrum occurs is known as λ_max.

Materials and Reagents:

1. UV-visible spectrophotometer

2. Standard quartz or silica cuvette (path length =1cm)

3. Colored and non-colored solutions

4. Buffer for blank

Procedure:

1. Switch on the spectrophotometer for few minutes to warm up and switch on the UV lamp.

2. Take two matched cuvettes. In the reagent blank cuvette add 3 ml of distilled water (Buffer). In the sample cuvette add noncolored compound to be tested.

3. Set the instrument at 200nm. Place the reagent blank cuvette in the holder and again adjust it to zero absorbance (or 100% transmittance).

4. Check the zero and 100% transmission to make sure that the instrument is properly adjusted.

5. Determine the absorption of sample against the blank.

6. Now reset the instrument at 210nm with reagent blank and record reading of the sample. Repeat this step at interval of 10nm each upto 390 nm.

7. For colored compounds start taking reading from 410 nm to upto 700 nm wavelengths on the tungsten lamp switching off the deuterium lamp.

8. Draw a graph of absorbance versus the wavelength to obtain the λ_max of each colored and non-colored compound.

Observation:

The graph may looks like it.

Color of Visible Light

Color Wavelength, nm Filter color/Complementary color

Voilet 400-435 Yellow-Green

Blue 435-480 Yellow

Green-Blue 480-490 Orange

Blue-Green 490-500 Red

Green 500-560 Purple

Yellow-Green 560-580 Voilet

Yellow 580-595 Blue

Orange 595-610 Blue-green

Red 610-750 Green-blue

(NOTE: blue absorbing solution appears yellow or green absorbing solution appears purple)

NOTE : Standard solution and their respective wavelength (λ_max)

Standard solutions Wavelength (λ_max) in nm

Protein solution 280

Nucleic acid 260

Bradford’s method 595

Biuret test 670

Folin Lower’s method 660

Ninhydrin method 570

Experiment no. 16

Verification of validity of Beer’s law and determination of the molar extinction

Coefficient of BSA (Bovine Serum Albumin)

Objectives:

1. To verify the validity of Beer’s Law

2. To determine the molar extinction coefficient of BSA

Principle:

Qualitative colorimetric estimations are based on two laws i.e. Lambert’s law and Beer’s law. Lambert’s law defines the relationship between the lengths of light path through the solution (The rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of light). While Beer’s law states that the intensity of a beam of monochromatic light decreases exponentially with the increase in concentration of the absorbing substance arithmetically. Combining the above two statement gives the Lambert-beer law ad states that the rate of decrease of intensity of light depends on the concentration and thickness of the medium and can be express by the equation:

Where A= absorbance

ε_λ = molar absorptivity (L mol^-1 cm^-1)

d= path length of the sample (cm)

c= concentration of the sample in solution (mol L^-1)

Absorbance is directly proportional to the other parameters, as long as the law is obeyed. After certain limitation the law is not obeyed and the straight line deviates from the normal in extreme cases of the concentration of samples and is called deviation of the law. The experiment is designed to explain why, for measurements made with samples of the same thickness d, the transmittance T of a sample decreases exponentially with increasing concentration c of the absorbing substance.

The Molar absorption coefficient, molar extinction coefficient, or molar absorptivity, is a measurement of how strongly a chemical species absorbs light at a given wavelength. It is an intrinsic property of the species; the actual absorbance, A, of a sample is dependent on the pathlength l and the concentration c of the species via the Beer-Lambert law, A = εcl.

Materials and Reagents:

1. UV-visible spectrophotometer
2. Standard quartz or silica cuvette (path length =1cm)
3. Phosphate buffer
4. Bradford reagent
5. BSA solution

Procedure:

1. Switch on the spectrophotometer for few minutes to warm up and switch on the UV lamp.

2. Take two matched cuvettes. In the reagent blank cuvette add 3 ml of distilled water (Buffer). In the sample cuvette add noncolored compound to be tested.

3. Set the instrument at 200 nm. Place the reagent blank cuvette in the holder and again adjust it to zero absorbance (or 100% transmittance).

4. Check the zero and 100% transmission to make sure that the instrument is properly adjusted.

5. Determine the absorption of sample against the blank.

6. Now reset the instrument at 210nm with reagent blank and record reading of the sample. Repeat this step at interval of 10nm each upto 390 nm.

7. For colored compounds start taking reading from 410 nm to upto 700 nm wavelengths on the tungsten lamp switching off the deuterium lamp.

8. Draw a graph of absorbance versus the wavelength to obtain the λ_max of each colored and non-colored compound.

9. For the determination of Molar extinction coefficient of BSA, Prepare BSA solution of concentration 2 mg/ml in both dH₂O and 0.9% NaCl.

10. Take the absorbance of BSA solution at 280 nm wavelength and calculate the E_percent.

11. Again calculate the molar extinction coefficient.

Calculation:

Table-1: Protein quantification by the Bradford method: protocol for preparing the standard curve and samples

Tube	BSA 10 mg/ml (ml)	H2O (ml)	BSA (mg/tube)	BSA (mg/ml)	Bradford (ml)
Blank	-	2.0	-	-	3.0
1	0.1	1.9	2.0	1.0	3.0
2	0.2	1.8	4.0	2.0	3.0
3	0.4	1.6	8.0	4.0	3.0
4	0.6	1.4	12.0	6.0	3.0
5	0.8	1.2	16.0	8.0	3.0
6	1.0	1.0	20.0	10.0	3.0
Samples	2.0				3.0

Note: Standards and samples should be analyzed in triplicate and processed at the same time, using same bath of reagents and assay conditions. Samples may require dilutions to fit in the range of absorbance values of the standard curve.

For Calculation of molar extinction coefficient

(ε_percent x c x L) / 10 = A

Where: c- concentration

L- pathlength (1 cm)

E_percent- percent solution extinction coefficients

A- Absorbance

(ε_molar) 10 = (ε_percent) × (molecular weight of protein)

Observation:

Standard values

Molecular weight of BSA = 66400

ε_percent = 6.6

ε_molar = 43,824 L M^-1cm^-1

References:

1. Blackshear P. J. (1984) Systems for polyacrylamide gel electrophoresis. In Methods in enzymology (Jakoby WB eds.) vol 104: 237-255.

2. Wilson K. and Walker J. (2002) Practical Biochemistry Principles and techniques, fifth edition, Cambridge University Press.

3. Sawhney S. K. and Sigh R. (2000) Introductory Practical Biochemistry. Narosa publishing house, New Dehli.

4. Gill, S.C. and von Hippel, P.H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319- 26.

5. Gel filtration principles and methods Amersham Biosciences.

6. Walker J.M. (2002) The protein protocols handbook, second edition, Humana press.

7. Dennison C. (2002) A guide to protein isolatioin, Kluwer academic publishers, New york.

8. Chatwal G. R. and Anand S. K. (2001) Spectroscopy Atomic and Molecular Fifth revised and enlarged edition, Himalaya Publishing House, Mumbai.

9. Plummer D. (1987) An introduction to Practical Biochemistry, Third edition.

10. Shrestha et. al; Delta Endotoxin Immunocrossreactivity of Bacillus thuringiensis isolates collected from Khumbu Base Camp of Mount Everest Region (2006) J. Food Sci. & Technol. Nepal 2:128-131.