Bcl-2 Detection in Western Blotting

Abstract: Bcl-2(B-Cell Leukemia/Lymphoma 2) is the key anti-apoptotic protein, and can maintain cell survival via inhibiting apoptosis. Bcl-2 family includes over 20 kinds of anti-apoptotic and pro-apoptotic proteins. Overexpression of Bcl-2 is closely related to the development and resistance to medical therapy of various cancers. Important roles of Bcl-2 in cancer research and cells show its potential target in cancer treatment. Steps of western blotting technique for Bcl-2 detection are specified below.

Keywords: BCL-2 Western Blot, Protein Sample Preparation, Method of Western Blotting, Selection of Suitable Antibodies

1. Preparation of Protein Samples

Successful protein sample preparation is very important for western blot, ensuring the protein integrity, high abundance and fully exposed antigenic epitope. If expression of the target protein in samples is very low or unavailable, the band can be hardly detected. Investigation of expression of the target protein in databases(e.g. BioGPS or Protein Atlas) before the experiment is necessary. E.g. Bcl-2 protein is just highly expressed in a few cell lines. Overexpression or induction can improve the level of lowly expressed samples.

2. Western Blot Optimization

1) Transfer: Wet transfer at 4℃ is suggested(constant voltage: 70V, 30min - 3h). Transfer duration for low molecular proteins is shorter and 0.22 µm membrane can be used. Extend transfer duration for macromolecular proteins to 3h. Add 0.1% SDS into transfer buffer. Reduce the concentration of methanol to 5%-10%.

2) Blocking: Block for 1h at room temperature with 5% skimmed milk powder or TBST. The blocking effect of skimmed milk powder is usually better than BSA.

3) Washing: Wash the membrane with 1×TBST three times for 5min each.

4) Antibody dilution: Dilute primary antibody following the ratio specified in the manual. Dilute secondary antibody with 5% skimmed milk powder or TBST.

3. Selection of Suitable Antibodies

Notes for antibody selection: Confirm whether antibodies are validated for WB. Cited antibodies are priority choice. Check whether the host species of antibodies matches with secondary antibodies. Besides, immunogen region(e.g. N terminal, C terminal or full length) should be the same as epitope of the target protein. Select high specific and sensitive antibodies based on expression level of the target protein in the sample. Judge via databases like Protein Atlas, CiteAb etc.

4. Conclusion

Expression abundance of proteins and antigenic epitopes recognized by antibodies are different. Details in protein preparation and WB procedure affects whether the experiment can be smoothly performed.

5. Recommended Products

REFERENCES

[1]Hyperglycemia-induced oxidative stress exacerbates mitochondrial apoptosis damage to cochlear stria vascularis pericytes via the ROS-mediated Bcl-2/CytC/AIF pathway, PMID: 39092597.
[2]Morin inhibits ubiquitination degradation of BCL-2 associated agonist of cell death and synergizes with BCL-2 inhibitor in gastric cancer cells, PMID: 40319008.