I have protein extracts from samples of testicular tissue from Wistar Rats, that were lyophilized contained RIPA buffer with protease inhibitors (150 mmol NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.8 mmol EDTA, 1.0 μg/mL aprotinin, 1.0 μg/mL leupeptin, and 35.0 μg/mL PMSF in 50 mmol Tris–HCl, pH 7.2). I’m using PBS at approximately pH 7.0, and it’s not completely reconstituting. I need it not to compromise the total protein dosage, for later Western Blotting and mas spectrometry analysis.
RIPA content is not applicable for mass spec proteomic assay...To eliminate the detergent content you should subject it to S-TRAP-, FASP like on membrane cleavage protocol...Thus, dissolve the content in a slightly alkaline buffer (usually ammonium bicarbonate) and add urea to improve solubility...For WB increase the added volume of your either phosphate or Tris buffer. If this does not work, during lyophilization, the protein content must be agglomerated owing to the concentration effect. Adding urea as a chaotropic agent, additional RIPA, or a mass-compatible detergent like DOC, rapigest may induce solubilization.
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