Hello everyone, I am working with samples derived from mice and rat models, and I’ve been having difficulty detecting BCL-2 (Cat.No: HY-P80565) consistently by western blotting. The signal often appears weak or unclear. Could anyone share optimized approaches for improving BCL-2 detection for it to work better in rodent tissues? Any advice on practical adjustments would be very helpful. Thanks!
Lucian Miles
Hi Fan,
- It is necessary to ensure that the cells themselves have a high expression level of BCL-2, and it is recommended to apply stimulation to further increase the expression level.
- During protein preparation, after adding lysis buffer on ice and lysing the cells, it is best to include a sonication step. Sonication helps in two ways: mechanically shearing DNA to prevent viscous gDNA from interfering with the protein lysate, and facilitating the full release of proteins from membrane structures.
- For blocking, primary antibody, and secondary antibody dilutions, use 5% skim milk. Compared with BSA, skim milk provides a better blocking effect.
The answer to this question is provided by MedChemExpress Technical Support.
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