Hi everyone
I made a fresh RIPA buffer using foll. composition
1) Tris Buffer 50mM (7.5 pH)
2)150mM NaCl
3)1%NP-40
4)0.5% Sod. deoxycholate
5)Protease inhibitor
After lysate was run on SDS-PAGE gel, i could not find lot of bands that i'd normally be able to visualise in the range of around 50-90 Kda when i used another RIPA buffer made a few months ago. Any suggestions or troubleshoot or changes that i should amend. Although i have run the lysates using the latest lysis buffer only once and will recheck it.
Another question is that which lysis buffer is the most appropriate for the membrane receptor proteins. Any published literature in this context would be highly appreciated.
THANK YOU
Dear Vikrant,
you may have several issues for not identifying the usual bands.
1. Your lysis buffer contains NP-40, which is a good detergent, but you can exchange it for 1% Tween-20, nevertheless;
2. Among protease inhibitors, does your buffer contains phosphatase inhibitors? I don't know which kind of protein you are looking for, but it is good practice for a lysis buffer to contain 1 mM of EGTA, 1 mM of sodium orthovanadate, 1 mM of sodium fluoride, 1 mM of PMSF, 50 ug of aprotinin and 50 ug of leupeptin (some of them may be in your cocktail, you may have to check). It is essential to ensure that your sample isn't degradated.
3. How are you lysing your material? Is it tissue or cells? You normally need to scrap cells and let them lyse in the buffer for some time, and then remove debris. For tissues, you first need to fragment it and then lyse it.
4. You shouldn't leave your lysate at -80 °C for long periods of time, because you may face protein degradation. You shouldn't submit your samples to many freeze-thaw cycles as well. It is better to denature it in a sample buffer and store at -20 °C.
5. Are you loading a good quantity of material in the gel? We normally have a very concentrated lysate and load about 100 ug of total protein.
We have seen several transmembrane proteins with the mentioned buffer. Perhaps other steps of the protocol may have experienced variability, leading to the effects you noted?
I would add 0.1% SDS in your buffer, it helps a lot to solubilize proteins, and is more efficient than non-ionic detergents like Triton or NP-40. If you are specifically concerned to obtain membrane proteins, you can directly employ 1%SDS hot solution by adding it directly to adherent cells (use it only if you don't need to perform IPs, subcellular fractionations and/or protein-protein interactions, in that case RIPA with 0,1%SDS is perfect).
Good luck
I think Eduardo Listik has given many great tips and suggestions that should help.
I used ready made `RIPA Lysis Buffer System: sc-24948` from santa cruz for both H9c2 cell line and a Rat model. I had a lot of samples to deal with in a short time so this cocktail can be made just before use. I think the steps 3-5 from Mr. Eduardo Listik is very important to get a good band.
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