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Removing phosphates


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#1 Gonzo

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Posted 10 December 2009 - 04:50 AM

Hi, there!

I would like to know if anybody has tried to remove phosphate postranslational modifications in proteins. Till now I just knew lambda phosphatase method. Moreover, I'm trying to use lysis bufffer without phosphatase inhibitors (NaF and Vanadate, for example). I think I read sometime ago something at this respect about EDTA. Maybe somebody knows an special buffer which impairs phosphorylation... Does anybody have an idea?

Thanks very much!

#2 laurequillo

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Posted 11 December 2009 - 02:53 AM

EDTA is known to inhibit metalloproteases. May also chelate ions that are co-factors of certain enzymes (For example If you are gonna use CIP you should avoid EDTA in your buffer).

Maybe you can try to use just PBS and NP40 (0.1-1%) (without any phosphatase inhibitor NaF, Sodium orthovanadate, EDTA...)

Edited by laurequillo, 11 December 2009 - 02:55 AM.

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#3 Gonzo

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Posted 11 December 2009 - 04:00 AM

Thanks, quiyo.

EDTA is known to inhibit metalloproteases. May also chelate ions that are co-factors of certain enzymes (For example If you are gonna use CIP you should avoid EDTA in your buffer).

Maybe you can try to use just PBS and NP40 (0.1-1%) (without any phosphatase inhibitor NaF, Sodium orthovanadate, EDTA...)






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