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Protein Electrophoresis:
Introduction, Selection Guide, Range Selection Guide

Introduction

Electrophoresis of proteins under native or denaturing conditions is one of the key methods used in proteomics. Under native, non-denaturing conditions, the electrophoretic separation of proteins depends on their size, shape and charge. The electrophoretic analysis under native conditions is therefore useful for applications in which the native properties of the protein are being investigated. The separation under denaturing conditions is mainly based on the molecular weight of the protein and this is therefore an important tool for assessing the purity of a sample and for providing an estimation of the molecular weight of the protein.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Nearly all proteins are soluble in the presence of the anionic detergent sodium dodecyl sulfate (SDS). SDS binds to proteins with high affinity (mass ratio is 1:1.4). This binding confers a negative charge to the polypeptide and destroys high order protein structures. The amount of the bound SDS is proportional to the size of the protein molecule, and the net charge of the denatured polypeptide becomes proportional to its size. The final position of protein band is a result of equilibrium between an electrostatic force and the braking force of friction between the gel matrix and the protein during the SDS/protein complex migration towards anode. SDS-PAGE is the most widely used method for characterization of proteins and evaluation of their molecular weights, as well as for establishing homogeneity of protein samples.
Tertiary and quaternary protein structures must be disrupted completely to achieve protein separation according to molecular weight. This is accomplished by reduction of the inter-molecular and intra-molecular disulfide bonds of polypeptides which are reduced by the addition of agents like dithiothreitol (DTT) or 2-mercaptoethanol to the protein samples. To enhance disruption of disulfide bonds, the protein samples are heated. All important components for the preparation of protein samples are included in the Fermentas Loading Buffer Pack.
Normally, SDS-PAGE is performed in the standard Tris-glycine-SDS electrophoresis buffer. For separation of small proteins and peptides, Tris-tricine-SDS electrophoresis buffer is recommended.

Native Protein Electrophoresis
Native structure and conformation is very important for biological protein functions such as enzymatic activity, interaction of antibodies with their ligands, etc. To preserve native protein structure and conformation, proteins are analyzed by non-denaturing electrophoresis. The pH of the buffer system is an important parameter for optimal protein separation. Native protein electrophoresis is often carried out in Tris-glycine electrophoresis buffer. Protein samples used for the native electrophoresis should be devoid of strong denaturants such as SDS. Neither Fermentas protein ladders/markers, nor the Protein Loading Buffer Pack are suitable for native protein electrophoresis as they contain SDS and DTT.

Estimation of Protein Molecular Weight
Protein SDS-PAGE is widely used as a data source for protein molecular weight determination. After digitizing a gel image, the standard curves are built based on the mobility of protein standards. The data of standard protein mobility is used to build a graph of relationship between the molecular weight of standard proteins and their relative mobility (Rf). Usually the functional relationship is calculated according to the formula log(MW) = a + b x Rf, where a and b are constants determined by calibration with known standards. MW of unknown protein is calculated by substituting its Rf in the above analytical expression. A new equation must be calculated for each gel, and data for several gels may be processed for the statistically robust results.
Fermentas offers both unstained protein standards and prestained protein standards for determination of protein molecular weight.
Prestained standards are convenient for monitoring the progress of electrophoresis, as well as the efficiency of protein transfer from a polyacrylamide gel to a membrane (Western blotting). Chromophores that are covalently coupled to the proteins of prestained standards affect their mobility. Therefore, prestained standards (PageRuler™ Prestained Protein Ladder, PageRuler™ Plus Prestained Protein Ladder and Prestained Protein Molecular Weight Marker) are recommended for approximate estimations. Each lot of prestained protein standard is calibrated against a precisely sized, unstained standard. Their calculated apparent molecular weights are reported in the Certificates of Analysis.
For precise determination of molecular weights, unstained protein standards (PageRuler™ Unstained Protein Ladder and Unstained Protein Molecular Weight Marker) should be used.

Discrepancies in Protein Mobility
Apparent molecular weights of Fermentas protein standards are calculated in Tris-glycine-SDS electrophoresis buffer. However, the bands of the protein standard may have different mobility in other electrophoresis buffer systems. The migration pattern of a particular protein standard in different buffers can be provided upon request.
Natively occurring protein modifications, like phosphorylation, glycosylation, etc, may alter protein mobility. The molecular weights of modified proteins may, or may not, correspond to those of unmodified standard proteins of the same size.

Protein Quantification
For protein quantification Fermentas offers Protein Standard Solution, a solution of BSA (bovine serum albumin) which is used as a standard for colorimetric determination of protein concentration by Lowry, Bradford or other methods.
Protein standards are suitable only for rough protein quantification on gels even though the approximate concentrations of each protein in the standard are provided in their descriptions.

Protein Detection
Proteins can be detected either within the polyacrylamide gel matrix or after their transfer to a polymeric membrane.
PageRuler™ Unstained Protein Ladder contains an integral Strep-tag® II sequence which can be detected directly on Western blots using Strep-Tactin®-AP conjugate or an antibody against Strep-tag® II.
Fermentas PageBlue™ Protein Staining Solution, based on Coomassie Brilliant Blue G-250 dye. This solution is recommended for highly sensitive and convenient protein staining on gels and PVDF membranes. PageBlue™ is extremely convenient as it does not require the destaining step. The detection limit of PageBlue™ is 5ng of protein. The PageBlue™ Protein Staining Solution is compatible with mass spectrometry, protein micro-sequencing and other applications.

Selection Guide

Fermentas offers protein standards for SDS-PAGE. The ready-to-use protein standards are available both in unstained and in prestained versions:


Analysis of PageRuler™ Unstained Protein Ladder:
A - using an Agilent 2100 bioanalyzer and Protein 200 Plus LabChip® Kit;
B - 4-20% Tris-glycine SDS-PAGE

Range Selection Guide

 

 

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Updated vasario 01, 2008 10:53