I am using the Roche cOmplete ULTRA Tablets for extraction of total soluble protein from leaf tissues. Does anybody know any cheaper but efficient one? Any suggestions would be greatly appreciated!
Cheapest option is to buy separate bottles of every protease listed in that cocktail tablet mixture - mix them and make concentrated stock by yourself. For safety, it is better to add some amount of cocktail tablet along with the mixture that you made. This could be the most efficient cost effective way that one can think of.
Just a note-
I will use only very authenticate vendor for inhibitor cocktail matter because this is one of the crucial step, cost dosent matter if you compromise here then things will fall apart.
Phenyl methyl sulphonyl flouride (PMSF) AT 5 mM works fine for inhibition of most serine proteases. Secondly, perform extraction at low temperature which shall keep most protease lowly active.
- 1 mM PMSF as inhibitor of Cys- and Ser-proteases is important, but in aqueous solution it is stable for only 20 min or so. Make a 1 M stock solution for example in i-propanol and add to your buffer immediately before use.
- Warning: do not use DMSO as solvent, as it makes the skin permeable for the highly toxic (AChE-inactivator!) PMSF. Less toxic variants of PMSF like AEBSF (trade name Pefablock) are available, but much more expensive (factor 30 or so).
- With Mdr1 I once found that PMSF alone wasn't sufficient to prevent degradation, but that aminocaproic acid (5 mM, another Ser-protease inhibitor) was also required. Thus different target proteins and matrices need to be taken into account.
- In addition, you may have to inhibit metal proteases (EDTA, EGTA), Cys- (E64) and Asp-proteases (pepstatin).
- IMHO a coctail of 5 mM aminocaproic acid, 0.1 mM each of EGTA and benzamidine (1 M stock in water), 1 µM each Leupeptin and Pepstatin (from 1 mM stock solutions in MeOH) makes a good starting point, with PMSF added as discussed above. The stocks (except EGTA, which can be kept at RT) are kept in the fridge, where they are stable.
- If you have an antibody to your protein do a western blot to check that you get only a single band, multiple bands indicate proteolysis or other post-translational modification. Shallow gradient gels are best for this, choose the %T so that your protein has a Rf of 0.4-0.6.
- Once you have done this, you can systematically check which of the above mentioned inhibitors is required in your case, leaving out the unecessary ones makes your buffer cheaper.
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