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Re: [ccp4bb] His tag does not bind. |
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CCP4bb navigationCCP4bb <-- 2007 <-- October 2007 <-- 11 October 2007Subject: Re: His tag does not bind. From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL Date: 2007-10-11 1. If the protein does not bind in a 'normal' buffer try first under denaturing conditions to see if everything is expressed correctly (a repeat of Artem's suggestion basically!). Protocols for that are in all materials manuals. 150 kD in E.coli is very very tough. 190 is even tougher. If you use a C-term tag, which has also the advantage it will select 'full length' proteins, take care of the first few codons; His- and other Tags are optimized for that. A silent mutation at position 4-5 (after the ATG) has made for us an easy 10-20 fold difference in the past. 2. Most buffer change pH depending on temperature. See for example: http://www.neb.com/nebecomm/tech_reference/general_data/tris_buffer.asp Always measure pH at the temperature you plan to work at, or make sure you always measure at the same temperature and be aware of the problem, look-up tables as above exist for many buffers. 3. For such a long protein an optimized-codon synthetic gene worked really well for us for a 100 kD protein, boosting expression 10-fold. consider it if it ends up to be low- yield trouble. A. On 11 Oct 2007, at 19:02, Jacob Keller wrote: > Recently, a make-or-break difference for me was making sure the pH > was what I thought it was: > > In the presence of 50mM HEPES pH 7.0 (at room temp), the pH of my > protein sample was actually > nearer to 6 at 4degC, as judged by pH paper. I am not sure whether > the pH difference was mainly > because of the temperature drop or because of other components in > the mixture, but adding an > additional 50mM TRIS pH 8.5 (RT) made all the difference. The pH > paper after this addition > indicated a pH of 7-8. In the former case, the protein was > completely undetectable in elution > fractions, and in the latter, was successfully purified. Of course > this makes complete biochemical > sense, but nevertheless I am very glad I checked the pH. > > Jacob Keller > > > On Wed, 10 Oct 2007 8:12:46 pm CDT changrui lu wrote: > > Dear all, > > I am trying to express a 150 kd protein in E coli. I have it in two > constructs, one with pmal-his and other with only his tag at N > terminus. The > full length protein can be detected both by sds and western using > anti-his > (190kd and 150kd respectively) but strangely neither binds to his- > column > very well. The majority of the full length comes through the column > either > at loading step or low salt wash step. The major species that gets > trapped > and eluted is the mbp-his truncation (~40kd). Some, though very > little, full > length protein did make it out the his column. The pmal-his > construct does > not bind amylose resin any better with majority flows right > through. All > purification are carried out under standard conditions as mentioned > in the > manuals. The protein is soluble and does not precipitate in the > columns. I > appreciate and ideas or explanations. > > Thanks in advance. > > Ray > Cornell Univerisity > ===========End of original message text=========== > > > > *********************************** > Jacob Keller > Northwestern University > 6541 N. Francisco #3 > Chicago IL 60645 > (847)491-2438 > j-keller2@northwestern.edu > *********************************** CCP4bb navigationCCP4bb <-- 2007 <-- October 2007 <-- 11 October 2007 |
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