I electroeluted protein into a buffer containing Glycine and tris base.Then concentrated it using centriprep tubes. Then I used a membrane cassete to dialyse it into PBS. A large amt of white powdery precipitate formed in the cassette. Any ideas why?
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#2
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Posted 20 May 2002 - 02:05 PM
Your protein is hydrophobic. To keep it in solution, you need Urea at 5 M.
Good Luck
Sam Brilliant
dstf@doctor.com
Ph.D. Rescue Team
A division of DSTF-Global
http://www.dstf.bigstep.com
#3
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Posted 24 May 2002 - 06:01 AM
Sam,
It all depends on what you need this protein for in the end. It will stay in solution in 5M Urea but the protein will be totally denatured and usable in functional assays. I have had this problem before and let me tell you it is often not trivial. The way a protein interacts with its solvent is a complex dance of many interactions, not only hydrophobicity. Definately read Protein Purification - Principles and Practice by R. K. Scopes. It is an excellent primer for beginning work with proteins.
It all depends on what you need this protein for in the end. It will stay in solution in 5M Urea but the protein will be totally denatured and usable in functional assays. I have had this problem before and let me tell you it is often not trivial. The way a protein interacts with its solvent is a complex dance of many interactions, not only hydrophobicity. Definately read Protein Purification - Principles and Practice by R. K. Scopes. It is an excellent primer for beginning work with proteins.
#4
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Posted 21 July 2004 - 01:05 AM
hai,
i think hydrophobicity of the protein is not a reason for precipitation after
dialysing against PBS because i too faced the same problem and get rid of
that by using low concentration of salt in PBS. this shows that the
white powdery precipitate may be due to incomplete removal of SDS and
then formation of KDS (potassium dodecyl sulphate) or SDS when dialysed against PBS
i think hydrophobicity of the protein is not a reason for precipitation after
dialysing against PBS because i too faced the same problem and get rid of
that by using low concentration of salt in PBS. this shows that the
white powdery precipitate may be due to incomplete removal of SDS and
then formation of KDS (potassium dodecyl sulphate) or SDS when dialysed against PBS
M. Naga Leelaram,
PhD student,
Indian Institute of Science,
Bangalore
India
PhD student,
Indian Institute of Science,
Bangalore
India
