[ccp4bb] Tev cleavage
Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
Re: [ccp4bb] Tev cleavage
Hey, I always had very good results with cleaving with TEV at 4C overnight (30min are to short at this temperature). Alternatively, I did the TEV cleavage at 16-23C for 1-3 h. 37C might just be to warm for your protein... Since some time I switched to the PreScission system instead of using TEV protease. This enzyme works better in my hands than the TEV protease. If you need a really fast and efficient cleavage (15min / 4C), you can use a HIS-SUMO tag which can be cleaved off using ULP1. This is the most efficient cleavage in short time that I know. Good luck! Karsten Am 02.05.2015 um 10:56 schrieb Giulliana Rangel: Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel -- Karsten Thierbach, Dr. rer. nat. California Institute of Technology Division of Chemistry Chemical Engineering Hoelz laboratory 1200 E. California Blvd., M/C 147-75 Pasadena, CA 91125, U.S.A.
Re: [ccp4bb] Tev cleavage
Giulliana, Alternatively to the previous excellent suggestions, you can add a few more residues between your protein and TEV-cleavage site. For example, if you have tag on the C-terminus you can add GSGS after the last residue: it should improve cleavage without significant impact on crystallization. Vitali From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Giulliana Rangel giulliana.ran...@gmail.com Sent: Saturday, May 2, 2015 12:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tev cleavage Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
Re: [ccp4bb] Tev cleavage
These are the usual culprits My buffers for cleavage and an on-column digestion worked good. (see below) Also most likely your TEV source (do not go cheap) enzyme is inactive (gone bad). Get a clone for TEV and make your own TEV in the lab. It save you a ton of money . 10 mM Tris-HCl (pH 8.0) 150 mM NaCl 0.1% IGEPAL CA-630 0.5 mM EDTA 1 mM DTT (add immediately before use from 1 M stock) Best wishes P On Sat, May 2, 2015 at 10:56 AM, Giulliana Rangel giulliana.ran...@gmail.com wrote: Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel -- P
Re: [ccp4bb] Alexander Rich passed away Monday April 27, 2015
On 05/02/2015 12:23 PM, Imre Berger wrote: Dear Edward - Would you be so kind and explain why you went ahead to post that comment about Alex Rich on CCP4, in a thread which announced the sad news of his passing away? Yes- I realized after posting it that it was inappropriate. If there is any way to remove a post, I will be glad to do so. In any case an apology is due. As for the explanation, I did not intend it to be in any way derogatory. I have never met Alex Rich, but Prof Sung-Hou Kim was my mentor in crystallography, and I have no doubt that their actions were completely honorable. As explained on the page I linked, it was all a misunderstanding based on poor communications between Kim and Rich, and rapid progress on the part of Kim that Rich was not aware of at the previous meeting. There was no evidence of actual misconduct on the part of Rich or Kim, as grudgingly acknowledged in the final letter from Cambridge. If only I had pointed that out in the email, instead of linking to that first accusatory message, it wouldn't have looked so bad. I had forgotten how inflammatory that first letter was! I was thinking this followed in the lines of Bob Sweet's post, that Rich was a hard-driving man and maybe not afraid of stepping on some peoples toes in order to achieve his goals. I never meant to imply misconduct, although after reading back on my post I can see that interpretation. My sincere apologies to the community and to the memory of professor Rich, Ed Berry I have checked your home page and your CV and it is not obvious to me at all what motivation or stake you could possibly have. Besides, knowing both Alex Rich and Aaron Klug and having discussed with them years ago, I think it is fair to say that only those two are concerned with the issue, and one of them has - very sadly - just died. In any case - in my view it is highly inappropriate indeed that you placed those comments on CCP4. Maybe you could be so kind and remove your contribution from the thread - it would be greatly appreciated. De mortuis nihil nisi bonum Imre -- Imre Berger PhD HDR Professor of Biochemistry Wellcome Trust Senior Investigator Coordinator, EC FP7 ComplexINC project The School of Biochemistry, University of Bristol UK The European Molecular Biology Laboratory EMBL imre.ber...@bristol.ac.uk iber...@embl.fr
