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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Polyclonal Antibody raising
Posted by: tarun895 (IP Hidden, New member, 1)
Date: April 14, 2005 06:46AM
Hello,
I have expressed a protein with His tag at N-terminal. I am using Qiagen Ni-NTA spin columnsto purify my protein under denaturing conditions. As my protein is in Insoluble fraction i have solubilised it in 8M Urea containing buffer as mentioned in their manual. Also I am doing the purification solely on the basis os pH. I hv not included any EDTA or Imidazole to the buffers. But I am facing a few problems... 1. Elution is not proper, ie a lots of protein is generally left bound to the column even if I am eliting it at a pH of 4.2 recommended is 4.5(wash at a pH of 5.4, recommended is 6.3) 2. Elution seems to be complete if I am eluting my protein in a larger volume ie around 1.5 ml per run per column. making my protein very very dilute. Real Problem 1: Right now i have purified around 2-3 mg of protein but its in an enormous volume of 65 ml...so the protein is very very dilute. I have use this for polyclonal antibody production but this diluted product can't be used as such also dialysis will b of little help considering such a huge and diluted amount. Can u please tell us some way out so that we can probably change the buffer from 8M urea to PBS and at the same time we can reduce the volume also to a significant amount may by 2-4 ml. 2: Again at the sametime I hv to use this protein for structural studies so i want to remove the His-Tag but am unable to find any specific clevage site...below is the sequence of our expressed protein with His-Tag. MGSSHHHHHHSSGLVPRGSH(<--to be removed) MTARAAGGSASRANEYADVPEMFRELVGLPAGSPEFQRHRDKIVQRCLPLADHIARRFEGRGEPRDDLIQVARVGLVNAAVRFDVKTGSDFVSFAVPTIMGEVRRHFRDNSWSVKVPRRLKELHLRLGTATADLSQRLGRAPSASELAAELGMDRAEVIEGLLAGSSYHTLSIDSGGGSDDDARAITDTLGDVDAGLDQIENREVLRPLLEALPERERTVLVLRFFDSMTQTQIAERVGISQMHVSRLLAKSLARLRDQLE If you can please suggest some of products to do that it will be of great help and i'll try to get it as soon as possible. 3: Also please tell me if i can reuse these columns for the same protein. How do i store them under what conditions. And in case I am using the column continuously that is after finish one round of purification if am directly goin for second round of purification with the same column what treatment is necessary to ensure proper binding and other steps. I will be really greatful to you if you could please help me out. As we are running out of time please try to be quick in ur reply. Hope to hear from someone soon.
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