Polyacrylamide Gel Electrophoresis for Western Blot

Western Blot is composed of polyacrylamide gel electrophoresis (PAGE), followed by an electrophoretic transfer onto a membrane (mostly PVDF or Nitrocellulose) and an immunostaining procedure to visualize a certain protein on the blot membrane. SDS-PAGE is a standard means for separating proteins according to their molecular weight. This technique includes one dimensional electrophoresis and two dimensional electrophoresis. One dimensional gel electrophoresis is used for separation of most routine proteins, and here we mainly describe this technique.

PAGE gel preparation for Western Blot

The first step of SDS-PAGE is gel preparation. SDS-PAGE gel formulas, including different percentages of SDS-PAGE resolving gel and stacking gel as well, are provided as below.

Rule of thumb:

• The smaller the size of the target protein, the higher the percentage of resolving gel; The bigger the size of the target protein, the lower the percentage of resolving gel.
• After adding TEMED and ammonium persulfate (APS), your gel will polymerize fairly quickly, so do not add these until you are surely ready to pour.

Recipe for Western Blot resolving gel solution

Gel (%) H2O (mL) 30% Acrylamide/
Bis-acrylamide (mL)
1.5 M Tris-HCl,
pH 8.8 (mL)
20% SDS (mL)* 10% APS (mL)* TEMED (mL)*
7 15.3 6.9 7.5 0.15 0.15 0.02
10 12.3 9.9 7.5 0.15 0.15 0.02
12 10.2 12.0 7.5 0.15 0.15 0.02
13 7.2 15.0 7.5 0.15 0.15 0.02

Recipe for Western Blot stacking gel solution (4% Acrylamide)

Gel (%) H2O (mL) 30% Acrylamide/
Bis-acrylamide (mL)
1.5 M Tris-HCl,
pH 8.8 (mL)
20% SDS (mL)* 10% APS (mL)* TEMED (mL)*
4 0.67 6.9 1.25 0.025 0.025 0.005
*, SDS: Sodium dodecyl sulfate; APS: Ammonium Persulfate; TEMED: Tetramethylethylenediamine.

Different types of polyacrylamide gel electrophoresis (PAGE)

According to experiment conditions, polyacrylamide gel electrophoresis (PAGE) can be divided into native PAGE and SDS-PAGE. For most routine Western Blottings, SDS and a reducing agent are also added to fully denature the protein and remove all higher order structure.

PAGE type Gel condition SDS* DTT or ß-ME* Notes
native PAGE native - - The proteins remain higher order structure
SDS-PAGE denaturing & reducing + + The proteins are removed all high order structure, including disulfide bonds
*, SDS: Sodium dodecyl sulfate; DTT: dithiothreitol; ß-ME: β-mercaptoethanol.

Positive controls for Western Blot

Commonly, positive controls are cell lines or tissue samples exhibiting expression of the target protein. Purified recombinant proteins can also be served as positive controls. Positive controls are used to prove that your Western Blot protocol has no issues. If the positive control lysate produces no positive signal, it means you should optimize the protocol.

Visit our "Cell Lysates" Page and choose your own postitive controls for your experiments.

Negative controls for Western Blot

Negative controls are lysates from samples known to not express the protein of interest. Similarly, negative controls are crucial to avoid nonspecific binding (false negative results) during Western Blotting procedure.

Knockdown or knockout tissue / cell lines are commonly used as negative controls.

Loading controls for Western Blot

Loading control antibodies are used as tools to ensure equal loading of a gel during western blot and are helpful when compare the western blot results qualitatively or quantitatively. Loading control antibodies are against loading controls which are usually encoded by housekeeping genes and express stably at high levels in many cell lines and tissues. Some common loading control antibodies include Actin, Beta Actin, COX IV, GAPDH, Histone H3, Alpha Tubulin and Beta Tubulin, etc.

Sino Biological offers reliable loading control antibodies with molecular weight from 16 kDa ~ 75 kDa, for the detection of whole-cell proteins, cytoplasmic proteins, mitochondrial proteins and nucleoproteins. In addition, we also offer Transferrin antibody which can be used as loading control in serum sample.

If you want to know more, please visit "Loading Control Antibodies for Western Blotting" Page.