protein dephosphorylation by alkaline phosphatase? - (Nov/09/2005 )

hellow everyone: I want to do protein dephosphorylation. I have alkaline phospharase in hand (Takara), but the the product instruction said it 'used for DNA dephosphorylation'. The technical assistant said it can be used for protein dephosphorylation but she don't know how to use!
Who has the protocol for protein dephosphorylation using alkaline phospharase? would you please give me some help? Thank you very much!

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I have the same task. Just I bought SAP from Promega. Exactly, all commercial APs are for DNA treatment. But Promega helped me with references of people who used the AP and got good results. I'm going to try dephosphorylation in one week. Until that I have no my own experience.
Ask Takara for references.
Good luck.
Mikki

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Should work the same.

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Thanks Mikki and Cole for your kindly help!

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Hello, you send this message at the end of 2005 and I wonder if you get an answer on your question. If so, where can I order the alkaline phospharase enzyme and can you send me the protocol for protein dephosphorylation? I hope you will reply on this message.
Already thanks.

Greetings YDerks

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has anyone ever worked with lambda-phosphatase? i used CIAP until today, but it does not dephosphorylate tyrosine residues.

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usage of Alkaline or Acidic phosphatses may not dephosphorylate all Y/S/T-phosphoryls of a protein as they are not really phosphoprotein phosphatases; you may have to think of more specific dephosphorylation; try a look at Stratagene´s brochure:

http://www.stratagene.com/lit_items/Dephos...R63US_08_05.pdf

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Hi
I also want to dephosphorylate my protein, I read in NEB protocols that it's possible to do that by CIP. by I don't know it's exact protcol,
does any one know that?


form NEB website:
Dephosphorylating Proteins with CIP

Calf Intestinal Alkaline Phosphatase (CIP) can be used to release phosphate groups from phosphorylated tyrosine, serine and threonine residues in proteins.
The following information can be used as suggested initial conditions for dephosphorylation of proteins with CIP.
10 units of CIP … in 60 minutes at 37°C in a 50 µl reaction. The concentration of phospho-P is 10 µM with respect to phosphate.
If the source of phosphorylated protein is a crude extract of cells or tissue it is very important to use the appropriate proteinase inhibitors in the lysis buffer (because 50 mM EDTA inactivates CIP by 100%) and to use short incubation times for dephosphorylation.
Inhibition: 10 mM sodium orthovanadate inhibits CIP activity (10 units) by 90%

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the protocol from NEB website recommends to re-suspend proteins in a reaction buffer to get protein concentration about 0.5-1.0 ug/10 uL (0.05-0.1 mg/mL) and then treat with CIP (0.5-1.0 U/ug protein). I would like to treat larger quantities of proteins (600-1000 mg) with AP, which would mean to work with 6-10 mL. Does anybody know if it is really necessary to keep the protein concentration as low as 0.1 mg/mL for treatment with AP, or could AP work with less diluted sample, e.g. 1 mg/mL?

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