[ccp4bb] Detergents to eliminate non specific aggregations
Hi, everyone, I can get my protein complex but there are some non-specific aggregation from the NMR spectra, and chaps can improve it. So, besides chaps, is there any other detergents to be used during crystal screening? All suggestions are welcome. ThanksRegards, Yuan
Re: [ccp4bb] Detergents to eliminate non specific aggregations
Hampton makes a Detergent Screen that works the same way as the Additive Screen. http://hamptonresearch.com/product_detail.aspx?cid=1sid=39pid=31 It has 96 unique detergent reagents for crystal screening. It is a bit expensive, but if you don't want to purchase the whole kit you could just download the formulation table and then you will have a nice long list of possibilities. Mike - Original Message - From: 商元 shangyuan5...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Saturday, August 27, 2011 4:55:27 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Detergents to eliminate non specific aggregations Hi, everyone, I can get my protein complex but there are some non-specific aggregation from the NMR spectra, and chaps can improve it. So, besides chaps, is there any other detergents to be used during crystal screening? All suggestions are welcome. ThanksRegards, Yuan -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
[ccp4bb] detergents
Hi all, I have been trying to purify cytosolic fraction of membrane protein whose domain boundries are unknown. hence I have made a series of deletion constructs. The expression and purification is not a problem. I get good yields of the proteins. But on a gelfiltration column, they run in the void volume, which suggest that they are huge aggregates. I have tried different salts, DTT bME etc but no change. I use sarkosyl in my lysis buffer. But could any one tell me if I can use any detergents as (bOD) in the purification protocol, and how much should I use. with regards Anita
[ccp4bb] Detergents
Hi, I had set up crystallization with a bicine as buffer and peg 400 as precipitant. I used the detergent DDAO/LDAO as an additive to the crystallization drop (one of the hampton additive screen condition, it says 5% on the vial) I have a clear drop and in the centre there is a shiny precipitate (looks like granules attached to eah other). Then I opened the drop to touch those granules with a needle, but suprisingly its like a skin and the skin surrounded my needle, and then some how I was able to but back the skin in the ppt. orelse the drop is clear if I remove the skin from the drop. I couldnot see the same on the control drop with no protein and just buffer. Does anyone have any experience with such kind of of shiny skins on crystallization drops?? Is it protein? Can I go forward and use it for seeding?? Please suggest with regards Rashmi
Re: [ccp4bb] Detergents
Rashmi, Yes, you should use the needle for seeding. The first thing to do is to seed your control drop to make sure that the needles are not bicine, as this buffer can give needles at certain pH at high PEG400 concentrations. Next (or at the same time) also seed other protein drops that you have already set up under similar conditions. I recommend streak seeding, so that even if crystal growth is slow you will see the streak line early on. Enrico. On Fri, 29 Apr 2011 12:43:21 +0200, anita p crystals...@gmail.com wrote: Hi, I had set up crystallization with a bicine as buffer and peg 400 as precipitant. I used the detergent DDAO/LDAO as an additive to the crystallization drop (one of the hampton additive screen condition, it says 5% on the vial) I have a clear drop and in the centre there is a shiny precipitate (looks like granules attached to eah other). Then I opened the drop to touch those granules with a needle, but suprisingly its like a skin and the skin surrounded my needle, and then some how I was able to but back the skin in the ppt. orelse the drop is clear if I remove the skin from the drop. I couldnot see the same on the control drop with no protein and just buffer. Does anyone have any experience with such kind of of shiny skins on crystallization drops?? Is it protein? Can I go forward and use it for seeding?? Please suggest with regards Rashmi -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
