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Insoluble Protein purification

His-tag Protein Pur Insoluble Protein Sarkosyl

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#1 APP_Biolab

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Posted 02 February 2012 - 01:25 PM

Hi

First que - what is the difference between insoluble protein and inclusion bodies?

Second ques - I am trying to purify some insoluble Protein having the His-tag. My protein size is 30 kDA. I have tried the small scale 50 ml expression test where I soaked/resuspended my cell pellet in the Lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM Imidazole, 5% of Sarkosyl, pH-8.0). I diluted my Supernatant from 5% to 0.3% sarkosyl in the final volume and loaded to the Ni-NTA Spin column Qiagen (b'coz of the compatibility issue with Ni Column). The small scale test looks promising on gel and I was able to solubilize 50% of my insoluble protein. I can see a clear band in supernatant which has 50% intensity if I consider the Crude Cell lysate as 100%. However, not able to purify in the final stage. (It might not bind to the column) I am planning to repeat the same thing with big scale expression of 6 litre.

Does anyone provide me any detailed protocol for the His-tag protein expression and purification along with Srakosyl in buffer (Native condition)? I am not sure how much Supernatant should I load to the big Ni column (HiTrap Chelating 5 ml× 2 GE Health Care). I might have to dilute it from 5% to 1% as the big column is compatibile with 1% sarkosyl. I am not sure how much protease inhibitor and DNAse need to be added to my Cell pellet before I start purification ?

#2 BIO5

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Posted 10 February 2012 - 08:43 AM

Hi, I am having the same problems like you purifiyng an insoluble protein (120 kDa) and I want it in native form too to make activity assays. Recently I found an article with some explanations about inclusion bodies and insolubiliy; and I am going to prove their methods. I post it here, so you can read and find a solution too:
1. http://www.ncbi.nlm....pubmed/20078429
2. http://www.jbc.org/c.../17980.full.pdf




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