[ccp4bb] protein turns brown

Dear all, I have purified protein from E.coli. expression system. the protein has been purified with three independant columns. Now during concentration step using amicon, the protein shows brown colour. what could be the reason. best regards and Thanks, sandy

Re: [ccp4bb] protein turns brown

sometime it does happen becoz of protein aggregation, or reducing environment. but it may be your protein color as well that visible during concentration     Vikrant       From: sandeep toskgu...@rediffmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, 24

Re: [ccp4bb] protein turns brown

Hi Sandy, Is there any possibility of your protein to contain an iron-sulfur cluster? Best Ricardo

Re: [ccp4bb] protein turns brown

Hi Sandy, If your protein is soluble (i.e. we're not dealing with brown precipitate), it is possible that the brown color is due to a bound iron-sulfur cluster. In this case, your protein was always brown, but you couldn't see the color when it was very dilute. As a quick test, you could

Re: [ccp4bb] protein turns brown

Maybe you should give us a hint about the identity of your protein (if you dare;-)). I'm sure there are folks around who may be able to say whether or not your protein is supposed to be brown. You can't expect too much help if you don't provide (m)any details. Cheers, Bert On 9/24/10

Re: [ccp4bb] protein turns brown

[FeS] clluster ? Or some metal bound to your protein ? Is it dark brown or yellowish brown ? What's your protein concentration ? 25 mg/ml ? Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research

Re: [ccp4bb] protein turns brown

It could be caused by iron contamination in one of your buffers. We used to buy glycerol in a metal canister and metal would leach into the glycerol. Because of this, one protein that I worked with would turn yellow, even at relatively low concentrations. I did not have this issue when using

[ccp4bb] Fw: [ccp4bb] protein turns brown

Yellow or brown tinge is very common especially if you have used sonication to lyse the cells. It helps to wash the cells with buffer or even high salt and spin them down again before lysis. If you want brown then you should see what happens when you work with yeast. Probably flavins or some

Re: [ccp4bb] protein turns brown

and possibly the opposite to reduction which is oxidation - do you have cys residues? Perhaps your DTT or TCEP got exhausted? Remember you can add up to 10mM (not the traditional token 1mM). But remember to neutralize TCEP. Other possibility is adventitious metals as these give strong charge

Re: [ccp4bb] protein turns brown

Hi All, I also have a similar observation for proteins purified by Ni-NTA column. After concentrating the sample eluted from the Ni-NTA column, I see a brownish-yellowish tinge closer to the bottom of the filter with colorless buffer on top. This is observed even for non-metal binding and

Re: [ccp4bb] protein turns brown

I also have a similar observation for proteins purified by Ni-NTA column. After concentrating the sample eluted from the Ni-NTA column, I see a brownish-yellowish tinge closer to the bottom of the filter with colorless buffer on top. This is observed even for non-metal binding and non-FeS

Re: [ccp4bb] protein turns brown

That's a possibility. Do you know if the same coloration would arise if there is TCEP instead of DTT or b-ME? I usually have TCEP as the reducing agent. Thanks Fillip. Cheers, Shiva --- On Fri, 9/24/10, Filip Van Petegem filip.vanpete...@gmail.com wrote: From: Filip Van Petegem

Re: [ccp4bb] protein turns brown

According to Pierce TCEP is more tolerant of nickel and cobalt. However, TCEP is inactivated by other metals, namely copper, magnesium, silver and zinc. Dan Daniel A. Bonsor, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472 USA

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