Problem with protein purification

Use this category for questions regarding problems manipulating proteins in molecular biology applications (expression, detection, etc.)

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Problem with protein purification

Postby sapsr » Apr 21 2015 2:55 pm

I'm being able to purify my GFP. I'm using BL21 with pEXP5-NT/CALML3 (I removed the CALML3 and ad the GPF from a pVAX1-GFP).
I've tried to purify by IMAC and HIC but neither worked. In IMAC my protein didn't bind with the matrix and in HIC lots of proteins binded. :cry:
I didn't do any pre-purification- just sonication and centrifugation.


Can you help me? I have no idea what to do. :cry:


Thanks!
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Re: Problem with protein purification

Postby mdfenko » Apr 22 2015 7:06 am

you can download various, useful, free handbooks (including "Recombinant Protein Purification") from the ge lifesciences website.

they should help you figure out what to use to purify your protein.
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Re: Problem with protein purification

Postby sapsr » May 06 2015 3:59 pm

Than you so much for your help.

I optimized the production (lowered the growth temperature to 30ÂșC) and I was able to purify the protein with Ni Sepharose 6 Fast Flow, but now I have another problem!
I have two bands in my gel, one with the size expected and other in less 5kda... :? What could it be?
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Re: Problem with protein purification

Postby mdfenko » May 07 2015 6:56 am

how does the second band compare in staining intensity with the expected band?

is it stained for protein or specifically for your protein (western blot)?
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Re: Problem with protein purification

Postby chance.lee » Oct 27 2015 3:23 am

Suggest to do western blot, and see if the two bands are signal
1, If only one band with a signal, then the other is a miscellaneous band, you can:
Increase the concentration of sodium chloride to reduce non-specific binding, or check whether the bacteria were contaminated or not .
2, If the two bands both have a signal:
One may be that the protein expression is degraded. You should operate at low temperature or add a protease inhibitor.
Another possibility may be that the label is broken, which may be that the tags are too big, such as GST, MBP, or C terminal is not easily fully expressed, or the temperature is too high.
Last edited by mdfenko on Oct 27 2015 6:51 am, edited 1 time in total.
Reason: turned off signature link and deleted link to service within text
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Re: Problem with protein purification

Postby Chloe Mica » Nov 04 2015 3:25 am

I have experienced the same issue. I have tried the second method above and it worked. Maybe I should often visit the forum. I believe my lab level will get improved a lot. So many experts here.
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Re: Problem with protein purification

Postby NancyLee523 » Oct 27 2016 3:08 am

Hi,
Here is a link of Strategies for Native Protein and Recombinant Protein Purification . Hope it can help you.
http://www.biologicscorp.com/protein-expression-purification/strategies-for-native-protein-and-recombinant-protein-purification.html
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