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Gel Electrophoresis

Protein gel electrophoresis. Gel electrophoresis chamber for running protein gels

Protein gel electrophoresis is a common technique used to separate proteins for purification, characterization, and expression analysis. In this approach, charged protein molecules are transported through a gel by an electrical field. Their mobility through the electric field is dependent on protein size, shape, and charge. 

Both polyacrylamide and agarose gel matrices can be used in protein electrophoresis. These matrices serve as a sieve, allowing smaller proteins to travel more rapidly than larger proteins. Agarose has a large pore size and can be used to separate proteins with radius larger than 5-10 nm, such as large protein complexes. Polyacrylamide has a smaller pore size, can separate proteins ranging in size from 5 kDa to 2,000 kDa, and is most commonly used in protein electrophoresis.



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mPAGE™ color protein standards for SDS-PAGE and Western blotting applications
Protein Markers

Pre-stained and unstained molecular weight markers for protein gel electrophoresis, SDS-PAGE, and Western blotting applications.

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mPAGE™ bis-tris gels for protein electrophoresis
Protein Electrophoresis Gels & Buffers

Precast polyacrylamide protein gels, running buffers, and hand-casting reagents for hand-pouring polyacrylamide gels for PAGE and SDS-PAGE protein gel electrophoresis.

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Protein gel stained with Coomassie blue stain
Protein Electrophoresis Gel Stains

High sensitivity colorimetric and fluorescent protein gel stains, fixing solutions, and de-staining reagents for polyacrylamide gels, agarose gels, and PVDF and nitrocellulose membranes.

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Protein gel electrophoresis using the Millipore® mPAGE™ gel electrophoresis system
Electrophoresis Systems & Transfer Equipment

Electrophoresis tanks, blotting systems, and power supplies for protein gel electrophoresis and wet and semi-dry transfer.

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Several methods of protein gel electrophoresis exist, each method providing distinct information for proteins of interest:

SDS-PAGE

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) enables protein separation based on molecular mass. In this method, SDS detergent is incorporated into the running buffer. The SDS imparts a net negative charge on proteins, masking their intrinsic charge. As proteins are separated in the presence of SDS and denaturing reagents, they become less globular and more linear. As a result, the rate at which SDS-bound proteins migrate through the gel is primarily dependent on their size, enabling estimation of molecule weight by comparing to protein standards.

Native PAGE

In native polyacrylamide gel electophoresis, proteins are separated in a way that preserves their native conformation (tertiary structure), subunit interactions (quaternary structure and protein-protein interactions), and biological activity. In this method, proteins are prepared and run under non-reducing, non-denaturing conditions. Protein mobility is determined by a complex combination of factors, as each protein can migrate towards either electrode depending on its charge, and at a rate dependent on its shape and binding behavior. For this reason, native PAGE is not recommended for molecular weight determination. Native PAGE is typically used in applications that require purification of active protein, or detection by an antibody that only recognizes the native form of the protein.

Isoelectric Focusing (IEF)

Isoelectric focusing uses both an electrical field and a pH gradient to separate proteins by their native isoelectric point (pI). As proteins move through the pH gradient, their net charge changes. Under an electric field, each protein migrates to the pH where its net charge is zero (termed as the isoelectric point of the protein). During the separation process, proteins in the sample accumulate, or "focus", in specific and predictable locations in the gel. Isoelectric focusing is used in protein identification from complex samples (e.g., cell and tissue lysates, plasma), analysis of post-translational modifications, and separation of samples for mass spectrometry analysis.

2D PAGE

Two-dimensional polyacrylamide gel electrophoresis enables resolution of protein according to both its intrinsic isolectric point (pI) and mass. Separation by pI occurs through isoelectirc focusing (IEF). Separation by mass occurs via SDS-PAGE. 2-D PAGE provides the greatest resolution for protein analysis, and is commonly used in proteomic research to resolve hundreds to thousands of proteins on a single gel.

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