Hello,
I was doing limited proteolysis of a protein to purify certain domain and i have optimized with 500:1 of protein to protease (trypsin).
i took 2000ug(6mg/ml) (333ul) protein and 4 ug trypsin(0.5mg/ml) (8ul) and incubated at 22 degree Celsius for 30 min.
then i taken out the sample and as i added 40 ul PMSF ( 100mM stock) for ceasing further proteolysis, immediately the protein precipitated down (clotted)
i am not able to understand, why this happened.
please suggest me protocol for limited proteolysis?
PMSF as serine inhibitor is usually used in a final concentration of a range of 0.1-1.0 mM. and as you know it does not dissolve in water, when it you prepare it, first of all you should dissolve in very small amount (few drops) of ethanol then complete the volume with water. The other thing is it is unstable & its half life is short & depends on the pH of the solution it is 110 min. at pH 7,&= to 55 min at pH 7.5 While it is 35 min. at pH 8 after that it will degrade .May be what is happen with you is the pro-teases present in your solution continue to work & what you obtain is the product of this process .The other possibility is you are using too much of PMSF which is insoluble.& of course you can check if your protein is in the aggregate or in the solution by running conventional electrophoresis to look where is your protein. Good luck
Hello Dr. Raghu Ram,
stock concentration of PMSF was 100 mM (dissolved in mathenol) and final concentration in protein enzyme mixture 12 mM.
before PMSF addition, protein didn't show any precipitation and after PMSF whole solution became cloudy.
I think you are using too much PMSF,repeat your exp. by using the correct final concentration of this inhibitor,Meantime reread my first answer
thank you very much hasan, i will repeat with low concentration of PMSF.
May be try to use a water-soluble trypsin inhibitor, AEBSF. A I recall the working concentrations are around 0.1mM, depending on trypsin concentration.
You would have added enough methanol to make it up to 10%. That could have precipitated the protein especially since the methanol would be even more concentrated where it meets your protein solution. at what I call “hot spots” unless you added slowly and mixed very well while adding. Two possible alternatives. You can use half to ten times less PMSF. Or, you can substitute with water soluble AEBSF. I usually use a final concentration of 5 mM. The usual precautions about instability especially in alkaline solution applies.
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