Re: [ccp4bb] crystallization of a weird protein
try to change ur buffer instead of PBS u can use Tris or HEPES. In PBS buffer u will get salt crystals in many condition becoz of phopshaphate try to use nonionic detergents in ur soluton .. On Fri, May 20, 2011 at 8:29 PM, Nian Huang wrote: > It might be SDS precipitation. Although 0.1% SDS is generally considered > not high enough to precipitate at 4 degree, it might interact with other > components in your solution to form even less soluble material. > > Nian > > > On Fri, May 20, 2011 at 9:36 AM, WEI MIN wrote: > >> Dear All >> >> I have a difficulty to crystallize my membrane protein(his tagged). I got >> the salt crystals from many different screening conditions. The protein is >> in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree >> although it forms the milk solution. >> The solution is getting clear in the room temperature within 5 mins when I >> set up the trays. >> >> I would like to take any advice. I would appreciate your input. >> >> Best >> >> Min >> > > -- Vandana Kukshal Postdoc Fellow structure biology group international Center For Genetic Engineering and Biotechnolgy India
Re: [ccp4bb] crystallization of a weird protein
It might be SDS precipitation. Although 0.1% SDS is generally considered not high enough to precipitate at 4 degree, it might interact with other components in your solution to form even less soluble material. Nian On Fri, May 20, 2011 at 9:36 AM, WEI MIN wrote: > Dear All > > I have a difficulty to crystallize my membrane protein(his tagged). I got > the salt crystals from many different screening conditions. The protein is > in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree > although it forms the milk solution. > The solution is getting clear in the room temperature within 5 mins when I > set up the trays. > > I would like to take any advice. I would appreciate your input. > > Best > > Min >
Re: [ccp4bb] crystallization of a weird protein
Hi Wei, this milk is precipitated/microcrystals of SDS probably with some phosphate since you are in PBS buffer. I would have two suggestions. 1-Can't you find a better detergent than SDS for your membrane protein? Have you run a detergent screen for this protein to find a milder and more crystallization friendly detergent for the reconstiution/purification and handling of your sample. SDS is very very rarely used for membrane protein crystallization. 2- I would try to avoid preparing a protein for crystallization in a phosphate containing buffer (unless you have no choice). Phosphates tend to yield more salt crystals at the screening stage. Hope this helps. > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 356 Boyer Hall office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] crystallization of a weird protein
The "milk" solution is a suspension of lots of micro-crystals which form when temperature goes down. When you take the solution out, it worms up and the crystals dissolve. If you take a look at them under high magnification microscope in the cold room, chances are you'll see those crystals. I cannot give any recommendations on chemical makeup of your solution but you could try using the temperature gradient as the driving force for crystallization. Good luck, Nukri Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of WEI MIN Sent: Friday, May 20, 2011 9:36 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization of a weird protein Dear All I have a difficulty to crystallize my membrane protein(his tagged). I got the salt crystals from many different screening conditions. The protein is in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree although it forms the milk solution. The solution is getting clear in the room temperature within 5 mins when I set up the trays. I would like to take any advice. I would appreciate your input. Best Min
[ccp4bb] crystallization of a weird protein
Dear All I have a difficulty to crystallize my membrane protein(his tagged). I got the salt crystals from many different screening conditions. The protein is in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree although it forms the milk solution. The solution is getting clear in the room temperature within 5 mins when I set up the trays. I would like to take any advice. I would appreciate your input. Best Min
