I suspect the protein I'm trying to detect is sensitive to SDS in my lysis buffer.
Of course, I could shorten the lysis duration but it's not a good option for several reasons.
Does anyone have experiences with some different lysis buffers, specifically for:
- small size proteins cca 17kDa
- Drosophila brain sample
- western blotting
If you used something that worked well with small proteins and could share the recipe, I'd appreciate!!
Thank you in advance!
Hello Anja Krivograd
You could use a lysis buffer containing a mild non-ionic detergent and a combination of various salts and agents to enhance extraction and stability of proteins. NP-40 can be used to solubilize proteins. The buffer could also contain low salt concentration, usually 120 mM NaCl. Some salt is needed to prevent non-specific protein interactions. EDTA which is a common additive that has multiple functions including protease inhibition and protection against oxidative damage may also be added to a final concentration of 5mM. Tris is another additive that could be added to the lysis buffer which is used to buffer the pH and prevent protein denaturation. You could also add protease inhibitors in the lysis buffer prior to use in order to protect proteins.
The recipe of the lysis buffer is as follows.
NP-40/Triton X-100 lysis buffer:
50 mM Tris HCl, pH 8.5,
120 mM NaCl,
5mM EDTA,
1% detergent (NP40 / Triton X-100).
The concentration of salt and detergent could be altered according to your needs. Protease inhibitors should be added fresh to the lysis buffer at the time of lysis.
Try this lysis buffer. Hope it works!
Best.
What exactly makes you think that your protein is "sensitive to SDS"? Are you having problems with aggregation, degradation etc? Some more information is needed!
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating