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Author: Subject: How to crystallise protein?
redwater18
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[*] posted on 2-2-2004 at 19:19
How to crystallise protein?


I am a beginner in the aspect of macromolecular structure.at the begining ,i meet some problems.
so need your hands to me.
Long time passed ,in the aspect of macro-molecular crystallization,i acquired nothing all but failures.who has some experiments in the crystallization of protein to give me a hand ,thanks?
At first , the question is how to chose the correct precipitant to crystal single-chain ,including 17 pairs disulfide and one sulfhydyl ,protein?


[Edited on 3-2-2004 by chemoleo]
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[*] posted on 2-2-2004 at 21:44
attention redwater18


Please do not start multiple threads to talk about one topic. Also, please use the "edit" button to change your original post if you have something more to say and nobody has yet replied to your message.



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chemoleo
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[*] posted on 3-2-2004 at 06:34
How to crystallise... it's an art :)


Ok first of all I merged your three posts into one, to make things simpler. Pay heed to Polverones suggestions! :)

To answer your question, tell me first what you have:
@How big is your protein? in kDa?
@How many amino acids?
@What's its pI, basic or acidic?
@it has 34 cysteines???? making 17 disulphide bonds???? That is a very large number.


Then:
@did you confirm your protein is folded?
@Did you confirm that the protein forms a single species, i.e. a single monomer or multimer, and not mixtures thereof?
@Did you confirm it doesn't form random aggregates, which maybe soluble but not crystallisable?
@Do you have any measure on the stability of the protein? I.e. what is its thermal/chemical denaturation point? How long can you keep it, i.e. in the fridge, before it loses it's activity (if it has any measurable one)? How about 17 deg C, the temperature employed in crystallography?
@How well does it behave if oxygen is present?
@Did you measure whether the S-S bonds really are present, or are they partially reduced (which may possibly lead to covalent random crosslinking, thus preventing crystallisation)
@Did you confirm that your protein is clean (i.e. by SDS-gel / mass spectrometry)?

To the crystal trials itself
@Did you do a screen? How many conditions did you employ? Hanging or sitting drop?
@What was your concentration of the protein? betw. 10-40 mg/ml?
@How long did you wait until you decided it doesn't work? Sometimes things take as long as 3 months to crystallise

Just answer those, and maybe we can help ya more :)

[Edited on 3-2-2004 by chemoleo]




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[*] posted on 3-2-2004 at 09:11


Just an interesting sidenote:

I found out that tannins in tea will precipitate proteins. That finally solved the question why I always got a very fine rag like precipitate when adding aspartame to hot tea.




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[*] posted on 4-2-2004 at 01:19
thanks


thanks .
thanks
wholely thank you to be here to answer my question .i have some questions,but for my English is poor ,so ....sad
but i will try my best to write clearly my question .welcome you to discuse my question.
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[*] posted on 4-2-2004 at 01:37
qu.


then,i want to ask whether the crystallization that it is easy to solve in it reservior is true crystallization? and it's resolution is rare
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chemoleo
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mad.gif posted on 4-2-2004 at 08:00


Redwater - DO NOT CROSSPOST! :mad:

What's the point of posting the same silly question many times? It won't get you more attention that way, instead people get pissed off with you and do not bother to reply.

Quote:

then,i want to ask whether the crystallization that it is easy to solve in it reservior is true crystallization? and it's resolution is rare


I don't understand what you mean. true crystallisation? Rare resolution??
If you actually are really asking something of substance, get someone to check your posts so we understand what you mean.




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[*] posted on 4-2-2004 at 08:50


Not understanding english very well is not a reason to crosspost even after several warnings!

You're banned from posting for a week.




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[*] posted on 9-4-2004 at 11:35
X-ray Crystallography


Where do you want to use it for ?

You can actually use Protein crystals to get some nice pictures about there structure!

To do this you need a Crystal, a source of X-rays and an X-ray sensitive sheet.

To get a nice Crystal you first must reduce the solubility of the protein of interest.
You could use AmmoniumSulphate to do this,other salts can be used too.

Its a very time-Consuming and patience-needing Thing to try.

Par Example'

3M ammoniumSulphate Added to Myoglobin[present in blood],after waiting for several days,gives nice Crystalls.

But what really is interesting is the next step-->

If you were to have an X-ray source[any ideas someone one were to get this,some 'cheap' minerals maybe??] you could make an actuall Photograph of the atomic Structure of the protein !!!


Only one beam of Wavelength 1.54 Armstrong-that is ;achieved by accelarating the present electrons onto a copper plat.....]
This beam is directed on to the Crystall.When it strikes, some of the beams diffract into different directions .
These difractions are detected by the photo-sensitve sheet[just an ordinary unprocessed photograph-roll]
You can then see on the sheet where the beams went and thus you can see the structure of the atom ,although some mathematics and computing must be used to get it really as a 3D picture ,THough the 2d picture should be very niCE too.

The X-ray source is a '?' though




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[*] posted on 9-4-2004 at 12:01


Whoops, totally forgot, I gave redwater18 his posting rights back now...



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