Electrophoresis
Please click on the links below to select the appropriate product selection guide:
Electrophoresis in Western blotting is the step by which proteins are separated on a polyacrylamide gel. This technique is known as Polyacrylamide Gel Electrophoresis or PAGE. The separation can be performed for preparative or analytical purposes and carried out either under native or denaturing conditions.
 | To monitor the protein progression through the gel, one or more wells are usually dedicated to molecular weight markers. These markers are a mixture of proteins where the molecular weight of each component is known.
Prestained molecular weight markers indicate migration of proteins with similar molecular weights. This is important for protein identification during the detection step. |
GE Healthcare offers a wide range of electrophoresis products for Western blotting.
Factors to consider in electrophoresis :
- Protein size and use of sodium dodecyl sulfate (SDS)
The principle of PAGE relies on the capacity for proteins to migrate through gel pores when placed under an electrical field. In general smaller proteins migrate faster through the gel pores than the larger ones. As proteins have different electrical charges that affect their mobility, sodium dodecyl sulfate (SDS or sodium lauryl sulfate) is usually added to protein samples and buffers. This confers a negative charge to all proteins, ensuring they will migrate toward the positively charged anode. SDS also breaks up aggregates and non-covalently bound multimers.
Resolution of the target protein on SDS-PAGE depends on the protein size and the porosity of the gel. Thus, one may have to test several acrylamide concentrations to optimize its migration conditions. Polyacrylamide gels are classified in two subclasses: homogenous and gradient gels. The former refers to a uniform polyacrylamide concentration throughout the gel. The latter refers to a linear progression of acrylamide concentrations from top to bottom, resulting in a wider separation range, but with less resolution.
Polyacrylamide gel composition is indicated by two different percentages: the total percentage of acrylamide (%T) and the amount of the crosslinker used (%C), which is usually 2.6%. The higher the %T is, the smaller the pore sizes are, and the more difficult it is for proteins to migrate through the gel.
A typical Western blotting setup uses two gels, one above the other: the stacking gel and the separation gel. The stacking gel is a homogenous gel with large pores (%T = 4 - 5%) used to concentrate the protein sample in a thinner space before it enters the separation gel. This increases the bands' resolution in the separation gel. In contrast, the separation gel is a stiffer gel where the %T can range from 5 to 20%, either homogenous or a linear gradient.
Use the chart below to estimate the % acrylamide required for the molecular weight of your target protein.
Target size range (kDa) | %T in separation gel |
36 – 205 | 5.0% |
24 – 205 | 7.5% |
14 – 205 | 10.0% |
14 – 66* | 12.5% |
14 – 45* | 15.0% |
|
*Larger proteins fail to move significantly into these gels
Find out how our Western blotting products are made to work together
See our excellent track record as a supplier for the research community
See our product selection for each Western blotting workflow step | 
