Peter Akers

Questions and Answers (5) View all

• Answer added in Protein Purification
  7 Protein forming insoluble precipitate during purification!
  By Peter Akers · University of Auckland
  Peter Akers · University of Auckland

  After. After concentration CaCl2 was added (to make a final concentration of 1mM) and the solution was incubated at 37deg for optimal protease activit... [more]

  After. After concentration CaCl2 was added (to make a final concentration of 1mM) and the solution was incubated at 37deg for optimal protease activity
  Following
• Answer added in Protein Purification
  7 Protein forming insoluble precipitate during purification!
  By Peter Akers · University of Auckland
  Peter Akers · University of Auckland

  Yes, the protein has been purified using the same protocols as I have used, however this time expression was much higher (i.e much more protein) so I ... [more]

  Yes, the protein has been purified using the same protocols as I have used, however this time expression was much higher (i.e much more protein) so I think this could be the issue. There is certainly a lot more protein in the precipitate than the supernantant. The last place it was soluble was during concentration at room temperature by diafiltration. It then precipitated once I added the protease in 50mM Tris, 1mM CaCl2 pH 8 and incubated at 37deg. Thanks for your help!
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• Question asked in Protein Purification
  7 Protein forming insoluble precipitate during purification!
  I was wondering if anyone could provide any wisdom that could help me with the following problem: in short, my protein of interest is forming insolubl... [more]
  I was wondering if anyone could provide any wisdom that could help me with the following problem: in short, my protein of interest is forming insoluble precipitates during proteolysis with EKMax protease. The reaction was done under almost standard conditions as listed in the EKMax manual (37deg, 50mM Tris pH 8, 1mM CaCl2 but no tween20). When I ran a SDS-PAGE gel I saw a big fat band corresponding to my protein in the precipitate and almost nothing in the supernatant, which is where protein normally is. My normal field is chemistry, so I don't know everything there is to know about what could go wrong. The purification does involve a rapid dilution step for refolding, and I am suspicious that something may have gone wrong at that stage, although nothing irregular was observed. My big fear is that the protein may not have folded correctly and may be lost forever. If anyone can give me any insight into what can cause proteins to form insoluble precipitates under such conditions I would be most appreciative, thanks! Ps. I can add images of gels from tomorrow if it helps
  By Peter Akers · University of Auckland
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• Answer added in Optimization of Growth Conditions
  5 Bacillus subtilis growth conditions
  By Peter Akers · University of Auckland
  Peter Akers · University of Auckland

  Thanks for the tip, I'll give it a try! 

  Thanks for the tip, I'll give it a try!
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• Question asked in Optimization of Growth Conditions
  5 Bacillus subtilis growth conditions
  Can anyone help me with B. subtilus growth conditions? I have tried LB with mixed results and cant find a clear answer in the literature. Can anyone s... [more]
  Can anyone help me with B. subtilus growth conditions? I have tried LB with mixed results and cant find a clear answer in the literature. Can anyone suggest a growth medium and temperature?
  By Peter Akers · University of Auckland
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