What conditions to apply for detection of low abundance proteins?
277 人阅读发布时间：2022-10-20 14:31
It is crucial to know as much as possible about the target protein, before starting experimental work with detection using Western blot. Why is that?
There are major differences in the protocol to apply, depending on the amount of target protein in the analyzed sample. For proteins of low expression, the following is recommended:
- Use the specific cellular fraction (for non-cytoplasmic proteins)
- Concentrate your sample using TCA/acetone precipitation
- Use the specific organ/tissue where the protein is most concentrated. You can check expression levels here
- Use a treatment that may increase the amount of target protein
- Use 6-8 M urea in the extraction and loading buffers (keeps proteins unfolded)
- For proteins that may be targets for proteasome degradation, use an appropriate inhibitor
For proteins of high abundance, other adjustments of the protocol should be applied. Check what Western blot conditions to apply for high-abundance proteins here.
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