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Problem with protein purification By FPLC


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#1 Xtal_ghai

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Posted 29 November 2010 - 04:12 AM

Hi all,

I am facing a problem with protein purification by FPLC. ALL of a sudden since past six months all my protein preps seems to give me a weird type of sharp peak in the FPLC trace rite before my protein elutes and when i run the fraction on the gel there is no protein in there and other fractions seems to have protein which is pure but still i seem to get this peak all the time. Sometimes i notice milky suspension in the fraction volume which disappears. I wondered if it was nucleotides so i chked for nucleotide conc in the eluted protein using nanodrop but dint get any reading suggesting no nucleotide content.

Can anyone please suggest what it could be.

Many thanks for your help.

Regards
Xtal_ghai

#2 BioMiha

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Posted 29 November 2010 - 05:18 AM

Hi,
The sharp peak before your sample might be an air bubble or something in your sample that is absorbing at 280 nm (acetone or cysteine). The milky suspension might be due to the precipitation of your protein in your elution buffer, which disappears if you dilute or dialyze your sample. What type of purification are you doing?

#3 Xtal_ghai

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Posted 29 November 2010 - 12:36 PM

Hi,
The sharp peak before your sample might be an air bubble or something in your sample that is absorbing at 280 nm (acetone or cysteine). The milky suspension might be due to the precipitation of your protein in your elution buffer, which disappears if you dilute or dialyze your sample. What type of purification are you doing?


Hi Many thanks for your protein. Exactly that is what i thought initially but then since past six months in each every purification i do and with different proteins as well, i get that peak. To get this rite if i am doing something wrong in injecting i made my colleagues to inject for me and they also get that peak in my pruifications. Regarding the precipitation i have tried centrifuging tht fraction at high speed but there are no signs of precipitation. The biggest strange thing is it always come along with my protein elution volume.

Regards
Rajesh

#4 Xtal_ghai

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Posted 29 November 2010 - 12:47 PM

Hi,
The sharp peak before your sample might be an air bubble or something in your sample that is absorbing at 280 nm (acetone or cysteine). The milky suspension might be due to the precipitation of your protein in your elution buffer, which disappears if you dilute or dialyze your sample. What type of purification are you doing?

Sorry forgot to mention that i always perform affinity chromatography as a first step utilizing the His tag of GST tag and then as a second step i perform gel filtration on FPLC.

Cheers
Rajesh

#5 BioMiha

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Posted 01 December 2010 - 04:26 AM

I am not sure if I understand correctly. The fraction, where you observe this sharp peak in your FPLC chromatogram, is free of all protein (or just your protein of interest), but still in this same fraction you observe a milky suspension? If you purify with affinity chromatography using the His tag, what if it is the imidazole that you use for elution that is giving you the peak? It can absorb A280nm. You say you checked this fraction on nano-drop. What kind of chromatogram did you get = are there any peaks at a certain wavelength or nothing at all?




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