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Protein precipitates out so fast


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5 replies to this topic

#1 shimshady

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Posted 09 April 2009 - 01:01 PM

I am expressing a human protien in Rosetta BL21 cells and i get great expression. Our sonicator has recently broken so for the mean time ive been doing freeze thaw cycles. Is this bad for the protein? Anyways i purify a whole lot of protein about 60 mgs or so. However the longer it sits out, it just precipitates, even after ive removed the imidazole (i use a Ni column). I know the protein is sort of stable because someone else has made it and it works fine. Is this a by product of the freeze thaw cycles? Thanks

Andrew

#2 shimshady

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Posted 10 April 2009 - 08:01 AM

Anyone think that the imidazole and my protein dont like each other? I wonder if can collect the eluent from the column into dilute buffer with maybe glycerol? Thoughts, concerns, comments?

#3 klinmed

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Posted 11 April 2009 - 02:17 AM

Anyone think that the imidazole and my protein dont like each other? I wonder if can collect the eluent from the column into dilute buffer with maybe glycerol? Thoughts, concerns, comments?


Good for you. Lovely levels of expression!
Think you may be on the right track with dilution/glycerol. Probably a good idea to do a simple stability trial. I really donīt think that the imidazole is the problem. Questions and suggestions:

1) Is your purified protein too concentrated? Try diluting to < 1 mg/ml.

2) Are you storing it too near itīs isoelectric point (where it is least soluble)? Try diluting into say 50 mM MES buffer pH 6.5 and 50 mM Tris 8.5.

3) As you suggest add glycerol to 10 - 20%. I have found that ethylene glycol (50%) is very good for storing proteins. It can prevent aggregation, is non-denaturing and allows you to store protein as a liquid at-20 oC. It is easily removed by dialysis or gel filtration.

4) Does your protein contain free thiols? If so, add 1 mM DTT to the storage buffer.

5) Does your protein not like high or low ionic strength? Try dialysis against 50 mM pH 6.5 and 8.5 buffer with and without 250 mM NaCl.

6) If hydrophobic interactions are a problem, and glycerol or ethylene glycol do not work, try using a low concentration of a non-ionic detergent such as Tween 20 or NP40. Usually as a last resort since they are almost impossible to remove. Ok though if their presence does not influence what you want to use your protein for.

7) In really tough cases, and when desperation sets in, try buffer additions such as arginine or 1 - 2 M urea. Not nice but....

8) Use a combination of some of the above.

Hope this helps.

#4 sudhakar mutyala

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Posted 21 April 2009 - 04:07 AM

Anyone think that the imidazole and my protein dont like each other? I wonder if can collect the eluent from the column into dilute buffer with maybe glycerol? Thoughts, concerns, comments?


Good for you. Lovely levels of expression!
Think you may be on the right track with dilution/glycerol. Probably a good idea to do a simple stability trial. I really donīt think that the imidazole is the problem. Questions and suggestions:

1) Is your purified protein too concentrated? Try diluting to < 1 mg/ml.

2) Are you storing it too near itīs isoelectric point (where it is least soluble)? Try diluting into say 50 mM MES buffer pH 6.5 and 50 mM Tris 8.5.

3) As you suggest add glycerol to 10 - 20%. I have found that ethylene glycol (50%) is very good for storing proteins. It can prevent aggregation, is non-denaturing and allows you to store protein as a liquid at-20 oC. It is easily removed by dialysis or gel filtration.

4) Does your protein contain free thiols? If so, add 1 mM DTT to the storage buffer.

5) Does your protein not like high or low ionic strength? Try dialysis against 50 mM pH 6.5 and 8.5 buffer with and without 250 mM NaCl.

6) If hydrophobic interactions are a problem, and glycerol or ethylene glycol do not work, try using a low concentration of a non-ionic detergent such as Tween 20 or NP40. Usually as a last resort since they are almost impossible to remove. Ok though if their presence does not influence what you want to use your protein for.

7) In really tough cases, and when desperation sets in, try buffer additions such as arginine or 1 - 2 M urea. Not nice but....

8) Use a combination of some of the above.

Hope this helps.


Hi

I completely agree with Klinmed,

But usually a minimum salt concentration (50-150mM NaCl) will help the protein to stay in its native state. Immidazole with high protein concentrations will accelerate the precipitation so dilute or diafilter the protein by using Tris Nacl or phosphate buffer. Hope this will work for you.

Yours
Sudhakar

#5 haguilar

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Posted 05 June 2009 - 02:32 PM

Hi guys, I don't mean to steal this thread but I figured there are already some relevant comments to the problem I'm having.

My biggest problem is protein precipitating out of my elution fractions after thawing. I perform the purification (His6) at room temp, collect my elution fractions, and then analyze small aliquots by Coomassie; meanwhile, I freeze the remainder of the elutions at -20 degrees. During my last purification, I wanted to attempt to purify away some residual contaminants by pooling the fractions containing my protein of interest, diluting them in a large volume of buffer of identical composition, then adjusting the Imidazole concentration to make the purification more stringent.

However, when I thawed my elution fractions, nearly all of them had visible precipitates. I added 1% Triton X-100, and sonicated the pooled fractions and that seemed to help a little bit but when I attempted to load this pool onto my Ni column it became clogged and useless.

The composition of my buffers for the original purification were as follows:


Lysis Buffer:
50 mM NaH2PO4 (pH 8.0)
300 mM NaCl
50 mM Imidazole
1% Triton X-100
+ protease inhibitors + lysozyme + DNAseI +RNAse

Wash Buffer (used 10 mL of this after loading with the sample)
50 mM NaH2PO4 (pH 8.0)
300 mM NaCl
50 mM Imidazole
1% Triton X-100

Elution buffers (collected 2x 1 mL fractions of increasing concentrations of imidazole):
50 mM NaH2PO4 (pH 8.0)
300 mM NaCl
Imidazole --> 75 mM, 100 mM, 125 mM, 150 mM, 200 mM
NO TRITON

could the problem be the omission of triton X-100 from the elution buffers? or is freezing/thawing not a good thing to do [what is the alternative?]? To answer some of the above questions - I don't believe I'm purifying a massive quantity of protein - the band is not really even discernible in my bacterial protein lysate, its only after concentrating with the Ni column that I can see a clear band (a good size, but certainly not 'massive'). Also, the PI for my protein is <6.0, so I think the pH of the buffers (8.0) is probably ok in terms of solubility.

Any help you can provide would be great, thanks!

H

#6 mdfenko

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Posted 08 June 2009 - 08:09 AM

freezing is your problem. some proteins denature when frozen, yours may be one of them.
if this is the problem then you can add glycerol to the solution to prevent it from freezing at -20C or store at 4C.

it is also possible that you are seeing the sodium phosphate crystallize. sodium phosphate doesn't like to be cold. if this is your problem then you may need to use a different buffer.
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