My RIPA cell lysis buffer - Western blot (Nov/09/2010 )
Yesterday I was making a modified RIPA cell lysis buffer and the recipe (per 100 ml) is as follows:
5 ml 1M Tris-HCL (pH7.4), 3 ml 5M NaCl, 1 ml 10% NP-40, 0.25 g sodium deoxycholate, 1 ml 0.5M EDTA, 0.2 ml 0.5M EGTA, and fill with ddH2O to reach 100 ml.
And sodium deoxycholate was added at last and was hard to dissolve. And then this morning I tried again with sodium deoxycholate dissolved in 75ml ddH2O first before everything else was added and no precipitation was produced.
So what I'm asking is that why sodium deoxycholate was hard to dissolve when it was added after every other ingridient was mixed.
Thanks a lot
Potentially yes. In general the solubility of different compounds is not influenced by other compounds in solution, but this is not the case when the solution is saturated or close to saturation with another compound. In your recipe nothing is close to saturation that I can see, but there may be some solubility issues from the presence of other detergents in the solution.
I tried to make some RIPA again yesterday. This time I first dissolved sodium deoxycholate in 75 ml ddH2O and then added solutions of Tris, NaCl, EDTA and EGTA. Finally 10% NP-40 was added without stirring, ddH2O was added to reach 100 ml, and the resulting mixture was stirred briefly. But this time there was no precipitation produced during the process.
So,the reasons why I screwed up before were that sodium deoxycholate requires a larger amount of solvent to dissolve and that I added it after everything else was mixed first (including 0.1 ml 100% NP-40).
Oh, my supervisor, who is an MD working in the same hospital where I work and a PhD student in Yang-Ming University Taiwan, told me that this RIPA recipe requires no protease inhibitor because even with the inhibitor protein samples still degraded fast and protein quatification and Western blot were conducted right after the samples were coolected. I don't know this was due to how he handled the samples or something else. So does someone have same problem?
Protease inhibitors are quite labile, so degrade reasonably fast in solution and don't stand freeze/thaw cycles very well. Some proteases are not inhibited by the common protease inhibitors (e.g. PMSF, orthovanadate) so it could be that your protein of interest is targetted by one of these proteases. Also, some tissues such as the pancreas and the gut are full of proteases so they swamp the conventional amounts of inhibitors used.
It still wouldn't hurt to add some protease inhibitor (cOmplete from Roche is a good mix and very convenient) as it will help with stability of other proteins such as actin that you might blot for.
Always keep your lysates on ice and thaw slowly on ice to help minimise degradation. Don't heat apart from the denaturing step for denaturing PAGE.