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Min, I forgot one more thing. If you try to grow crystall of membrane protein and get tiny crystal, you may try to add 5~10% IPA + 5mM NH4SO4 to addjuct the surface charge of your protein. The crystall may get larger. Folks like this trick. Good luck. Kevin On Tue, Mar 13, 2012 at 10:38 AM, Kevin Jin <[log in to unmask]> wrote: > 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 <[log in to unmask]> wrote: >> Thank you Kevin, >> >> I will try to remove b-ME as you suggested. >> >> Min-Kyu >> >> | -----Original Message----- >> | From: Kevin Jin [mailto:[log in to unmask]] >> | 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 <[log in to unmask]> >> wrote: >> | > Hi Kevin, >> | > >> | > Could you tell me more detail about beta-mercaptoethanol problem? >> | > >> | > Min-Kyu >> | > >> | > | -----Original Message----- >> | > | From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf >> | > Of >> | > | Kevin Jin >> | > | Sent: Monday, March 12, 2012 3:32 PM >> | > | To: [log in to unmask] >> | > | 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 >> | > | <[log in to unmask]> 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" <[log in to unmask]> >> | 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:[log in to unmask]] >> | > | >> | Sent: Monday, March 12, 2012 11:16 AM >> | > | >> | To: Min-Kyu Cho >> | > | >> | Cc: [log in to unmask] >> | > | >> | 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 >> | > | >> <[log in to unmask]> >> | > | >> 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 >>