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CCP4BB  March 2012

CCP4BB March 2012

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Subject:

Re: My protein precipitates at r.t and dissolves at 4 oC

From:

Kevin Jin <[log in to unmask]>

Reply-To:

Kevin Jin <[log in to unmask]>

Date:

Tue, 13 Mar 2012 11:19:56 -0700

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (192 lines)

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
>>

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