From: Min-Kyu Cho
Date: 12 March 2012 16:02
----------
From: Kevin Jin
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
----------
From: Min-Kyu Cho
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
----------
From: Lijun Liu
----------
From: Artem Evdokimov
Could be one of those weird behaviors displayed by detergents where cloud point anomalously changes with temperature...
Artem
----------
From: Kevin Jin
I remember I saw the similar problem caused by beta-mercaptoethanol.
Kevin
----------
From: Min-Kyu Cho
Hi Kevin,
Could you tell me more detail about beta-mercaptoethanol problem?
Min-Kyu
----------
From: Kevin Jin
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 wrote:
> Thank you Kevin,
>
> I will try to remove b-ME as you suggested.
> |
> | 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
> |
> |
> |
> |
----------
From: Kevin Jin
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
Date: 12 March 2012 16:02
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
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
----------
From: Kevin Jin
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
----------
From: Min-Kyu Cho
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
----------
From: Lijun Liu
Min-Kyu,
This sounds like to be a hydrophobicity ~ temperature issue (as Kevin pointed out, pH gets involved too).
If your protein is sensitive in this regards, it could form different oligomerization states at diff temp, associated
with diff solubility. You may want to crystallize it at diff temperatures.
Lijun
----------
From: Artem Evdokimov
Could be one of those weird behaviors displayed by detergents where cloud point anomalously changes with temperature...
Artem
----------
From: Kevin Jin
I remember I saw the similar problem caused by beta-mercaptoethanol.
Kevin
----------
From: Min-Kyu Cho
Hi Kevin,
Could you tell me more detail about beta-mercaptoethanol problem?
Min-Kyu
----------
From: Kevin Jin
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 wrote:
> Thank you Kevin,
>
> I will try to remove b-ME as you suggested.
>
> Min-Kyu
>
> | -----Original Message-----
> | From: Kevin Jin
> Min-Kyu
>
> | -----Original Message-----
> | From: Kevin Jin
> | 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,> | To: Min-Kyu Cho
> | Subject: Re: [ccp4bb] My protein precipitates at r.t and dissolves at 4
> | oC
> |
> |
> | 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
> |
> |
> |
> |
----------
From: Kevin Jin
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
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