Hi Min, Please try this way if you use your protein for crystallization.
1. collect the needle and run SDS page or FPLC to verify the presence of protein. Make sure it is not a buffer salt. 2. You don't need to do dialysis to remove b-ME, otherwise it will take too long and you may lose some protein. Here is what I did before: 1. Kee you stock protein solution with 2mM b-ME on ice-bath. 2. Use those eppendoff like tubue (~ 500ul) with membrane, which we use to concentrate protein with reasonable MW cut-off (I usually used 8KD). 3. Add protein solution with b-ME (2mM) in the tube, spin down (10K, eppendoff centrifuge) for 2mins, 4. Use your pipette to measure how much solution has been filted into the bottom tube. 5. add equal amount fresh buffer solution without b-ME to the top tube, Repeat 3~5 times, and make the fine concentration of b-ME to <0.5mM. Then use the protein immediately for crystallization. Actually, I used the same way for buffer exchange, instead of dialysis. Based on this way, I crystallized thiopurine methyltransferase with 2.0 Ang after folks' 5 years effort. The images is available here: http://www.jinkai.org/Crystal_imgs.html Best, Kevin On Tue, Mar 13, 2012 at 8:36 AM, Min-Kyu Cho <min-kyu....@live.com> wrote: > Thank you Kevin, > > I will try to remove b-ME as you suggested. > > Min-Kyu > > | -----Original Message----- > | From: Kevin Jin [mailto:kevin...@gmail.com] > | Sent: Monday, March 12, 2012 5:50 PM > | To: Min-Kyu Cho > | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4 > | oC > | > | Hi Min, > | > | I need to look back my note. Here is from my old memory: > | > | 1. The protein was loaded in FPLC column at 4 degree C and the collection > | was clear. In this case, i did not use Tris Buffer, 2. When the protein > | was warmed up in room temperature, ppt appeared. > | 3.In the buffer, only beta-mercaptoethanol problem was added, in addition > | to PBS buffer. > | > | In my case, I removed beta-mercaptoethanol just before assay and > | crystallization, it worked and no ppt. > | > | I could not remember the detail. > | > | I hope this would be helpful. > | > | Kevin > | > | > | > | > | On Mon, Mar 12, 2012 at 3:42 PM, Min-Kyu Cho <min-kyu....@live.com> > wrote: > | > Hi Kevin, > | > > | > Could you tell me more detail about beta-mercaptoethanol problem? > | > > | > Min-Kyu > | > > | > | -----Original Message----- > | > | From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf > | > Of > | > | Kevin Jin > | > | Sent: Monday, March 12, 2012 3:32 PM > | > | To: CCP4BB@JISCMAIL.AC.UK > | > | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves > | > at 4 > | > | oC > | > | > | > | I remember I saw the similar problem caused by beta-mercaptoethanol. > | > | > | > | > | > | Kevin > | > | > | > | > | > | On Mon, Mar 12, 2012 at 1:29 PM, Artem Evdokimov > | > | <artem.evdoki...@gmail.com> wrote: > | > | > Could be one of those weird behaviors displayed by detergents > | > where > | > | > cloud point anomalously changes with temperature... > | > | > > | > | > Artem > | > | > > | > | > On Mar 12, 2012 1:11 PM, "Min-Kyu Cho" <min-kyu....@live.com> > | wrote: > | > | >> > | > | >> I am using KPi buffer at pH 5.5, 100mM KCl, 2mM > | > beta-mercaptoethanol, > | > | >> 0.02% NaN3. > | > | >> > | > | >> Yes, I agree I should check CD melting curve to see temperature > | > | >> preference of my protein. > | > | >> > | > | >> Min-Kyu > | > | >> > | > | >> | -----Original Message----- > | > | >> | From: Kevin Jin [mailto:kevin...@gmail.com] > | > | >> | Sent: Monday, March 12, 2012 11:16 AM > | > | >> | To: Min-Kyu Cho > | > | >> | Cc: CCP4BB@jiscmail.ac.uk > | > | >> | Subject: Re: [ccp4bb] My protein precipitates at r.t and > | > dissolves > | > | >> at 4 > | > | >> | oC > | > | >> | > | > | >> | Which kind of buffer you use? If it is Tris, then temperature > | > | >> change will > | > | >> | cause pH change. > | > | >> | > | > | >> | Actually, this is a good way for crystallization. > | > | >> | > | > | >> | Kevin > | > | >> | > | > | >> | On Mon, Mar 12, 2012 at 9:02 AM, Min-Kyu Cho > | > | >> <min-kyu....@live.com> > | > | >> wrote: > | > | >> | > Hi all, > | > | >> | > > | > | >> | > I have a homotetrameric coiled-coil domain sample with 45aa > | > per > | > | each. > | > | >> | > While I store this sample at 4oC, the sample looks clear > | > w/o any > | > | >> | > particles. But when I took out the sample to my bench at > | > r.t, I > | > | >> can > | > | >> | > see there are precipitates (as stack of needle like > | > particles) > | > | >> at the > | > | >> | > bottom of the tube after several hours. Interestingly, when > | > I > | > | >> put it > | > | >> | > back into 4oC fridge, the precipitates disappeared and the > | > | >> solution > | > | >> | turned into clear again. > | > | >> | > > | > | >> | > Does anyone have knowledge of such behavior of any protein? > | > I > | > | >> | > appreciate any information related. > | > | >> | > > | > | >> | > Min-Kyu >
