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dialyse of a protein

protein purification

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4 replies to this topic

#1 NDUFB11

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Posted 11 December 2012 - 11:38 PM

Hello, I am a new member even though I know this this forum since long time ...
I have a question, I am trying to purify a protein of 11-12 Kda.
Using NTA-Ni Qiagen I have a lot of contaminant.
I know I could use other things for a better purification such as ionic exchange chromatography or something else( currently I don't have ) and I was thinking something else today, my idea is this:
my protein is 11-12 KDa, if I use the spin tube for Dialysis with a MWCO 30 KDa , do you guys think that I could collect my protein and keep the unspecific in the filter ?
And should I use a filter with a MWCO 10 or 30 ?

thank you

#2 HOYAJM

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Posted 12 December 2012 - 11:09 AM

I was having problems with contaminants after His-tagged purification and I tried many spin filters (microcon filters). In short, they dont work like you would think they should. I had many low MW contaminants and I used a 30 MWCO filter and most of them did not go through. So, you can give it a try as long as your proteins are much larger than 30Kd, you could even use the 10 MWCO filter as I am pretty confident your 11-12 Kda protein wont go through.

If you are getting a lot of contaminants, you should really consider optimizing your wash and elution buffers. By optimizing the imidazole concentration for these buffers I had great success eliminating contaminant proteins. Also, improper cell lysis led to more contaminants. These spin filters seem like an easy fix, but they just arent very effective.

#3 NDUFB11

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Posted 12 December 2012 - 01:57 PM

Thank you for your answer Enthusiast,
I will try soon and Il l let you know what happened, I tried to change different conditions such as different pH for the binding and washing, different concentration of imidazole either for the binding or for the washing but the results was to have less protein and less contaminants.
May I ask you what do you usually use for the cell lysis ?
what I do is to follow their protocol , plus protease inhibitor (PMSF) in the lysis buffer.
I don't use lysozyme and I go throw the sonication

#4 HOYAJM

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Posted 17 December 2012 - 12:08 PM

I use 1mg/mL lysozyme, add about 0.1-0.2% beta mercaptoethanol to my lysis buffer and then go through 25 minutes of sonication. I have noticed that if I freeze the pellets after expression, that the resuspension and lysis is much more complete. If you cannot get rid of the contaminants by optimizing the purification, then I think your best bet is to go for size-exclusion chromatography. It is expensive, but it is the best way to get a homogenous sample.

#5 NDUFB11

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Posted 18 December 2012 - 04:27 PM

I use to snap freeze the cells after the induction as well, otherwise the experiment would be too long just in one day.
I tried as I mentioned before to use the filter tubes with a MWCO of 30 kDa, finally the protein is pure, but the yield is about the 25%,
I am sure that changing something during the purification I can still have more protein.
Thank you hoyajm, I am new working with proteins and for sure I will have some more question in the future

have good holidays

-P

Edited by NDUFB11, 18 December 2012 - 04:28 PM.






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