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
>>
|