In standard Western blot (WB) approaches, denatured protein samples are separated according to their molecular weight with SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to a membrane. The analysis of different organs, cell-types, and subcellular fractions like membranes, versus cytosol or different organelles may also provide useful information about differential protein expression levels. Enhanced chemiluminescent (ECL) detection systems are very sensitive, but have a narrow linear detection range that can be used for protein quantification. In general, the experiment has to be carefully optimized for reliable results.
Important: Some proteins have special requirements for good separation (e.g. unboiled samples or special gel systems). Please refer to the remarks sections for western blotting on the respective data sheet.
Materials and reagents
- Ponceau S staining solution: 5% acetic acid, 0.1% Ponceau S
- 5% skimmed milk in Tris buffered saline with Tween 20 (5% skimmed milk-TBST): 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% (w/v) skimmed milk powder, 0.02% sodium azide, 0.1% Tween 20
- Washing solution A: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20
- Washing solution B: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl
- Substrate solution: Western Lightning® Plus-ECL PerkinElmer, Inc. or comparable product
Separate the protein sample to be examined and a molecular weight standard using SDS-PAGE and transfer to a nitrocellulose membrane by electro-blotting. Follow the manufacturer's instructions for your SDS-PAGE and blotting device.
- Stain the membrane with Ponceau S staining solution for several minutes at room temperature to check the efficiency of transfer.
- Rinse the membrane in water to remove the Ponceau S staining solution and incubate in 5% skimmed milk-TBST for 30 min on a lab shaker (gently rocking) at RT.
- Incubate in fresh 5% skimmed milk-TBST containing the primary antibody at the appropriate dilution and incubate for at least 2 h on a lab shaker at RT or overnight at 4°C.
- Wash 3-4 times with washing solution A for 10 min each time.
- Incubate in fresh 5% skimmed milk-TBST containing the recommended secondary antibody (anti-mouse IgG, anti-rabbit IgG, resp.) at the appropriate dilution and incubate for at least 1 h on a lab shaker at RT.
- Wash 3 times with washing solution A for 10 min each time.
- Replace washing solution A with washing solution B and let equilibrate for 5 min.
- Replace with fresh substrate solution and develop (X-ray film or ECL-reader). Exposure time can be shortened or extended, if signals are extremely strong or weak, resp.
A very weak signal may be caused by the primary and/or secondary antibody concentration being too high. The ECL substrate solution has a limited capacity, and high amounts of local peroxidase can use up all the substrate within seconds before the picture is taken in your ECL reader.
Please try a lower concentration of primary and secondary antibodies in this case.
Note: The SYSY standard protocol generates good results in the SYSY labs and may be used as a reference. However, to achieve the highest specific signal and lowest non-specific background signal, the best antibody concentration, incubation temperature, and incubation time must be determined individually.