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Nickel IMAC - How to avoid unwanted proteins?


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#1 thommyd87

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Posted 11 May 2009 - 09:01 AM

Hi! Im currently working with BL21-AI Competent Cells (Invitrogen) to express a his-tagged protein (expression-plasmid). I tried to purify the desired protein through a IMAC catridge (bioscale, immobilized nickel ion affinity chromatography) and successfully eluated my protein. Unfortunately the gel electrophoresis revealed that the eluate contains 2 additional (smaller) proteins. Does anybody got a clue where these come from, and how to avoid them?

Best greets from germany

Thommy

#2 swanny

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Posted 11 May 2009 - 06:12 PM

Hi! Im currently working with BL21-AI Competent Cells (Invitrogen) to express a his-tagged protein (expression-plasmid). I tried to purify the desired protein through a IMAC catridge (bioscale, immobilized nickel ion affinity chromatography) and successfully eluated my protein. Unfortunately the gel electrophoresis revealed that the eluate contains 2 additional (smaller) proteins. Does anybody got a clue where these come from, and how to avoid them?

Best greets from germany

Thommy

Hi.
What was your protocol? Did you have any imidazole in your bind/wash buffers? If t he other bands are significantly different in size to your protein, you can remove them by gel filtration, which is a good polishingstep.

Edited by swanny, 11 May 2009 - 06:13 PM.

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#3 T C

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Posted 11 May 2009 - 08:53 PM

Hey,

I also get these bands aroung 66 kDa. These are E.coli chaperones and whether they go or not entirely depends upon the target protein. I have found this helpful:

Use 500 mM NaCl and 5 mM imidazole in wash buffer and was extensively.

Further purify by anion exchange on FPLC.

Best,
TC

Hi! Im currently working with BL21-AI Competent Cells (Invitrogen) to express a his-tagged protein (expression-plasmid). I tried to purify the desired protein through a IMAC catridge (bioscale, immobilized nickel ion affinity chromatography) and successfully eluated my protein. Unfortunately the gel electrophoresis revealed that the eluate contains 2 additional (smaller) proteins. Does anybody got a clue where these come from, and how to avoid them?

Best greets from germany

Thommy



#4 chimaera

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Posted 12 May 2009 - 03:10 AM

Have you tried a Western blot?
There can be different reasons: premature translation termination (rare codon clusters can be a problem), uncut signal sequence, protein impurities and so on.

#5 thommyd87

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Posted 16 May 2009 - 03:14 AM

What was your protocol? Did you have any imidazole in your bind/wash buffers?


I did wash with 2 different (non denaturing) washing buffers: potassiumchloride 300 mM Monopotassiumphosphate 50mM and Imidazole 5 respectively 10 mM. Eluated with 250mM Imidazole.
Apart from this, is there any chance to assume, which proteins those bands (of ecoli) stand for? I mean u supposed they are chaperones (do they have many HIS?)


Have you tried a Western blot?


For what purpose? I used this eluate to immunise a rabbit. I then tried to get the antibodys through an NHS-activated collumn. Unhappily the westernblot didnt show any bands :(..

Edited by thommyd87, 16 May 2009 - 03:20 AM.





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