I am working to extract the whole protein from staphylococcus aureus. the steps include treating cell pallets with lysostaphine followed by water bath sonication. The collected supernatant is treated with acetone (4 times volume of supernatant)
The protein precipitated collected by centrifuge. So now the precipitate has solubility problem. I can not use any salts or SDS or urea for making it soluble as the sample to be sent for in solution digestion of proteins for mass spectrometry.
please suggest what would be better to sort out this problem?
Thanking you
Wash the pellet with 95% ethanol, air dry it, and then dissolve it in PBS, lysis buffer or directly to Laemmli buffer
you can find your answer here....
https://www.researchgate.net/post/Best_way_to_resuspend_Acetone-precipitated_protein_for_Western_Blot
best wishes
Depends on what you want to do:
For MS proteomics
1. Resuspend in 8 M urea, 50 mM Tris pH 7.5 - 8.0. Measure protein concentration using BCA. Reduce/Alkylate. Dilute atleast 8 times so that urea conc. is less than 1 M, Trypsinise, acidify with TFA and clean on C18.
2. You can also use 0.5% RapiGest in addition to Urea and Tris in the above solution. Remember to remove it before going for C18.
3. Resuspend in 4-5% SDS, 50 mM Tris pH 7.5-8.0. Save a portion for PAGE/WB, and other portion you can process with the FASP procedure.
I have used all three of methods, many many times, for whole cell protein precipitated from yeast cells. For large scale I recommend using method 2, and for small scale my favourite method 3.
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