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> 2-Dimensional Polyacrylamide Gel Electrophoresis

• How to Order
• Quality assurance
• Fees
• Sample Preparation
• Electrophoresis Procedures
• Image Analysis
• Protein Identification

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Proteins are resolved by two consecutive, high-resolution separations. The first is isoelectric focusing, which discriminates on the basis of isoelectric point. The second is SDS polyacrylamide gel electrophoresis, which discriminates on the basis of apparent molecular weight. Dyes are used to detect proteins following electrophoresis, and the intensity of staining provides a measure of protein abundance. The method derives from that first described by Patrick O’Farrell (O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 1975;250:4007-4021)

How to Order

Studies are discussed with investigators in advance so that experimental design can be planned in detail. N.B. Isoelectric focusing and SDS polyacrylamide gel electrophoresis are also offered as separate services that may be requested independently.

Quality Assurance

Experiments with replicate samples in the Hartwell Center indicate that most proteins present in excess of 1 ng show less than two-fold gel-to-gel variation in their measured abundance. However, because of the relatively large affects of biologically-based variation, the results of expression analyses should be confirmed by repeating experiments with independently harvested cell/tissue samples.

Fees

Please follow this link to a general fees page.

Sample preparation

Cells or tissues are lysed in a buffer containing 8 M urea, 2 M thiourea, 2% CHAPS, 0.3% (w/v) Bio-Lyte ampholytes from BioRad Laboratories, Hercules, CA., (usually pH range 3-10), 50 mM dithiothreitol, a trace of bromophenol blue, and a protease inhibitor cocktail (one mini tablet of Pefabloc SC, Roche Applied Science, Indianapolis, IN). This buffer:

• denatures and helps solubilize proteins with a wide range of physical properties, including membrane proteins,
• minimizes ionic strength in order to promote efficient resolution of proteins during isoelectric focusing,
• provides a reducing environment to inhibit oxidative processes,
• inhibits proteolytic degradation. (Additional inhibitors may be added as required, e.g. vanadate to inhibit protein phosphatases, or compounds to inhibit additional proteases.

N.B. Proteins should never be heated in the presence of urea. The reason is that cyanates, which accumulate in urea solutions, carbamylate primary amino groups on proteins at elevated temperatures, producing species with altered isoelectric point, changed susceptibility to proteases, and altered mass.

Investigators are asked to lyse cells/tissues in their own laboratory, using lysis buffer supplied by the Hartwell Center upon request. Prior to lysis, cultured cells should be washed with phosphate buffered saline or an equivalent protein-free buffer. The salt concentration should be minimized because elevated salt concentrations may significantly degrade separation during isoelectric focusing.

Protein quantitation should be performed in the investigator's laboratory prior to submitting samples to the Hartwell Center. Some methods for protein quantitation are sensitive to investigator's components of the lysis buffer. The method provided by Amersham Pharmacia Biotech as the PlusOne 2-D quantitation kit (Catalog # 80-6483-56) is compatible with the lysates, and is therefore recommended for the present application. For most samples, the number of spots visualized on a 17 cm wide 2-D gel is maximized with a protein load of 100-200 µg. On a 7 cm wide 2-D gel, a protein load of 50 µg is optimal.

Investigators are asked to perform protein precipitation with trichloroacetic acid to remove non-proteinaceous material that may interfere with isoelectric focusing. Precipitated protein is redissolved in lysis buffer at a concentration of 0.4-0.6 mg/ml for isoelectric focusing. This protein precipitation procedure provides excellent results from a wide variety of cell and tissue types.

Some protein sources contain a small number of proteins in disproportionately large amounts. Such samples are fractionated prior to electrophoresis to deplete the super-abundant species in order to increase the number of less abundant proteins that can be detected and quantitated. For example, human plasma or serum is treated to remove immunoglobulin G and albumin. Albumin is absorbed by passage through a POROS goat anti-human serum albumin cartridge from Applied Biosystems. The antiserum will also absorb serum albumin from cow, mouse, pig, rabbit and rat. IgG is absorbed with a POROS Protein G cartridge, also from Applied Biosystems.

Prior to isoelectric focusing, samples are centrifuged at 200,000 g for one hour at room temperature in a Beckman Coulter Airfuge CLS Ultracentrifuge to remove particulates that may interfere with protein separation.

An efficient protocol is available for consecutive extraction of RNA, DNA and protein from the same cell sample.

Electrophoresis Procedures

Isoelectric focusing is performed with immobilized pH gradient (IPG) strips from Bio-Rad Laboratories. Gradients of pH 3-10, 4-7, 3-6, 5-8 and 7-10 are available. Initial studies to check sample quality are performed on small gels that use strips of 7 cm length covering a pH range of 3-10. Samples are then subjected to detailed comparison using large format gels employing strips of 17 cm length. The pH range is chosen according to the requirements for each individual project. Commonly, pH 4-7 is a useful compromise between the two competing requirements: on the one hand to cover the largest possible pH range to maximize the number of proteins being screened, and on the other hand to maximize the separation between spots. In cases where higher resolution is needed, the narrow, overlapping ranges, 3-6, 5-8, and 7-10 may be employed.

IPG strips are re-hydrated by overnight incubation with sample at room temperature. The sample volume is 125 µl for a 7 cm strip or 300 µl for a 17 cm strip. Focusing is then performed in a Bio-Rad PROTEAN IEF Cell. The total focusing time is approximately 5 hours for a 7 cm strip, or 7 hours for a 17 cm strip.

Figure 1. Bio-Rad PROTEAN Isoelectric focusing apparatus

Prior to second-dimension separation, SDS-polyacrylamide gel electrophoresis, IPG strips are incubated with reducing agent, dithiothreitol, then with an alkylating reagent, iodoacetamide, to dissociate disulfide bridges and prevent further reaction of the resulting free cysteine side-chains. Strips are equilibrated in SDS buffer, and then laid on the top of an SDS slab gel 1 mm thick, where they are sealed in place with low-melting point agarose. The gel is normally made with 10% acrylamide. Electrophoresis is performed in an apparatus holding up to 12 gels (Bio-Rad PROTEAN Plus Dodeca Cell for 18 x 18 cm gels, or Mini-PROTEAN 3 Dodeca Cell for 10 x 8 cm gels). Separation is generally complete in 45 minutes for a the smaller gels, or in 4 hours for the larger gels.

Figure 2. Bio-Rad PROTEAN Plus Dodeca Cell (right), and the Mini-PROTEAN 3 Dodeca Cell

Gels are stained for protein with SYPRO Ruby dye from Molecular Probes Inc., Eugene OR. This is a fluorescent dye with sensitivity comparable to silver stain. Its principal advantages are simplicity of application and superior range of linear response. Gels are washed with water for 5 min, fixed with 10% methanol, 10% acetic acid, 80% water (v/v/v) for 30 min, and then stained for at least 3 hours with dye solution. Destaining is performed in fixing solution for 30 min. Sensitivity of detection is approximately 1 ng for protein spots after 2-D separation.

Image Analysis

Images of 2-D gels are acquired with a Fuji FLA-5000 laser scanner. 16-bit resolution is employed, and files are saved in .tif/.img format. Image analysis is performed using the Progenesis Workstation package from Nonlinear Dynamics Ltd., Newcastle-upon-Tyne, U.K. This package contains routines for automated spot detection, electropherogram matching, and comparative quantitation. Results of pairwise image comparisons in which one gel is designated as the reference and the other as the test sample are reported in terms of fold-increase/decrease in the volume of matched spots. The staining intensity of unmatched spots is also reported.

Figure 3. Comparison of a pair of bacterial cell strains. Isoelectric focusing was performed over the pH range 4-7 (acidic end on the left) A. Reference electropherogram - place cursor over the red box for a 3-D view. B. Sample electropherogram C. Warping vectors calculated for optimal matching of the two images. D. Difference map in which spots showing an increase of 3x or greater are highlighted in red, and those showing a decrease of 2x or greater are highlighted in green.

Figure 5. Animated superposition of images in Figure 3A and 3B. Aligned spot patterns are displayed alternately to facilitate discrimination of features that are different between the two gels.

Protein Identification

Preparation of Proteins for Mass Spectrometry
Proteins of interest are excised either manually, or with a ProPic Spot Picker from Genomic Solutions Inc, Ann Arbor, MI.

In-gel digestion is then performed with sequencing-grade, modified trypsin supplied frozen by Promega Corp., Madison WI. Digestion is performed either manually or automatically with a Proprep digestion station from Genomic Solutions.

Peptides released from gel plugs are then extracted, purified using C18 ZipTips from Millipore Corp., Bedford MA, and spotted onto MALDI targets for mass spectrometry. These operations may be performed either manually or with a Proprep spotting robot, from Genomic Solutions Inc.

 

Precautions Against Contamination
At all stages of post-electrophoretic sample processing, rigorous procedures are employed to minimize sample contamination. The most abundant contaminants are keratins, which derive from the operator. The precautions taken in the Hartwell Center to minimize keratin contamination include:

• Washing all surfaces contacting the sample or gels/gel plugs with 50% isopropanol, 40% water, 10% formic acid (v/v/v), and then 60% acetonitrile, 40% water, 0.1% trifluoroacetic acid (v/v/v) prior to use.
• Performing manipulations of samples and gels in a laminar flow or still air hood.
• Passing solutions through 0.22 mm filters.
• Purifying laboratory air by HEPA filtration.
• Wearing long-sleeved lab coat and latex gloves at all times when samples/gels are handled. N.B. latex gloves are washed to remove particulate material adhering to their outside surfaces after putting them on and prior to use.
• Employing robotic systems for sample handling whenever possible.

These precautions are sufficient to reduce keratin contamination to acceptable levels, even in samples in which the unknown protein is present at very low levels. This is a critical aspect of the methodology, because keratin contamination constitutes the primary limitation to the sensitivity of protein identification. The same procedures are employed for identification of proteins after one-dimensional SDS-polyacrylamide gel electrophoresis.

USE OF MASS SPECTROMETRY FOR PROTEIN IDENTIFICATION