2D-Gel Electrophoresis and Western Blotting

Two Dimensional Polyacrylamide Gel Electrophoresis

Why 2D GE?

• Oldest method for large scale protein separation (since 1975)

• Still most popular method for protein display and quantification

• Permits simultaneous detection, display, purification, identification, quantification

• Robust, increasingly reproducible, simple, cost effective, scalable & parallelizable

• Provides pI, MW, quantity

Steps in 2D GE

• Sample preparation

• Isoelectric focusing (first dimension)

• SDS-PAGE (second dimension)

• Visualization of proteins spots

• Identification of protein spots

1. Sample Preparation

• Sample preparation is key to successful 2D gel experiments

• Must break all non-covelent protein-protein, protein-DNA, protein-lipid interactions, disrupt S-S bonds

• Must prevent proteolysis, accidental phosphorylation, oxidation, cleavage, deamidation

• Must remove substances that might interfere with separation process such as salts, polar detergents (SDS), lipids, polysaccharides, nucleic acids

• Must try to keep proteins soluble during both phases of electrophoresis process

Protein Solubilization

• 8 M Urea (neutral chaotrope)

• 4% CHAPS (zwitterionic detergent)

• 2-20 mM Tris base (for buffering)

• 5-20 mM DTT (to reduce disulfides

• Carrier ampholytes or IPG buffer (up to 2% v/v) to enhance protein solubility and reduce charge-charge interactions

Other Considerations

• Further purification or separation?

• Subcellular fractionation

• Chromatographic separation

• Affinity purification

• Optimizing electrophoresis parameters

• IEF pH gradient, Acrylamide %, loading

• Limits of detection

• ng? (Coomasie stain) pg or fg? (Western)

2. Isoelectric Focusing

• Separation of basis of pI, not Mw

• Requires very high voltages (5000V)

• Requires a long period of time (10h)

• Presence of a pH gradient is critical

• Degree of resolution determined by slope of pH gradient and electric field strength

• Uses ampholytes to establish pH gradient

IEF Principles

Ampholytes vs. IPG

• Ampholytes are small, soluble, organic molecules with high buffering capacity near their pI (not characterized) • Used to create pH gradients via user • Gradients not stable • Batch-to-batch variation is problematic

• An immobilized pH gradient (IPG) is made by covalently integrating acrylamido buffer molecules into acrylamide matrix at time of gel casting • Stable gradients • Pre-made (at factory) • Simplified handling

Narrow-Range IPG Strips

3. SDS-PAGE

• Separation of basis of MW, not pI

• Requires modest voltages (200V)

• Requires a shorter period of time (2h)

• Presence of SDS is critical to disrupting structure and making mobility ~ 1/MW

• Degree of resolution determined by %acrylamide & electric field strength

• After IEF, the IPG strip is soaked in an equilibration buffer (50 mM Tris, pH 8.8, 2% SDS, 6M Urea, 30% glycerol, DTT, tracking dye)

• IPG strip is then placed on top of pre-cast SDS-PAGE gel and electric current applied

• This is equivalent to pipetting samples into SDS-PAGE wells (an infinite #)

4. Visualization of proteins spots

• Coomassie Stain (100 ng to 10 mg protein)

• Silver Stain (1 ng to 1 mg protein)

• Fluorescent (Sypro Ruby) Stain (1 ng & up)

5. Identification of protein spots

• Confirmed by Western, Northern or Southern

• Confirmed by amino acid composition

• Confirmed by amino acid sequencing

• Confirmed by Mw & pI

Advantages and Disadvantages of 2D GE

Western blot

The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette^[1] and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting.

· method of detecting specific proteins in a given sample of tissue homogenate or extract.

· uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions).

· The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein

· Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).

Steps in a Western blot

1. Tissue preparation

· Samples may be taken from whole tissue or from cell culture.

· A combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – can be used to separate different cell compartments and organelles.

2. Gel electrophoresis

· The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel.

· Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis

· It is also possible to use a two-dimensional (2-D) gel

3. Transfer

· Tranfer the proteins from the gel onto a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF by capillary action,

· Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane.

· "Blotting" process, the proteins are exposed on a thin surface layer for detection

· The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie

4. Blocking

· To prevent interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved.

· typically Bovine serum albumin (BSA) or non-fat dry milk are used

5. Detection

Two methods are used

5.1 Two step

Primary antibody

· Antibodies are generated when a host species or immune cell culture is exposed to the protein of interest (

· The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight.

Secondary antibody

· Exposed to Secondary antibody, directed at a species-specific portion of the primary antibody like "anti-mouse," "anti-goat," etc.

· The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase.

· Most commonly, a horseradish peroxidase-linked secondary is used in conjunction with a chemiluminescent agent, and the reaction product produces luminescence in proportion to the amount of protein.

5.2 One step

· This requires a probe antibody which both recognizes the protein of interest and contains a detectable label, probes which are often available for known protein tags.

6. Analysis

Diferent methods

6.1 Colorimetric detection

· This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane(such as peroxidase).

6.2 Chemiluminescence

· substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which captures a digital image of the Western blot.

6.3 Radioactive detection

· Placement of medical X-ray film directly against the Western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest

6.4 Fluorescent detection

· The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera