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Protein Phosphorylation


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#1 lia88

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Posted 02 February 2011 - 05:51 AM

Hi everyone,

I hope I'm in the right place to pose this question... I'm working with a transcription factor that we think needs to be activated by phosphorylation to form the active dimer. My question is, how do I phosphorylate my protein? It might be helpful to know that I've had a problem with keeping my protein soluble in the past (it's currently soluble now in 20% glycerol, 50mM Tris-HCl and 250mM NaCl) and that my end goal here is to study the protein-DNA interaction.

thanks in advance for any help!

#2 knuf

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Posted 02 February 2011 - 08:22 AM

It kind of depends on what all is known so far and some of it is just going to be trying and seeing what works. If you know the kinase that phosphorylates the protein you can do in vitro and/or in cyto phosphorylation and seeing if it is activated. If you know the site that is likely phosphorylated you can mutate the residue(s) to alanine(s), which cannot be phosphorylated. You can also do the reverse and mutate the residue(s) to aspartic acid, which have a negative charge and can sometimes mimic the phosphorylated state. It just depends on what you know, what you need to know, and what you have available to you.

#3 lia88

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Posted 02 February 2011 - 09:26 AM

It kind of depends on what all is known so far and some of it is just going to be trying and seeing what works. If you know the kinase that phosphorylates the protein you can do in vitro and/or in cyto phosphorylation and seeing if it is activated. If you know the site that is likely phosphorylated you can mutate the residue(s) to alanine(s), which cannot be phosphorylated. You can also do the reverse and mutate the residue(s) to aspartic acid, which have a negative charge and can sometimes mimic the phosphorylated state. It just depends on what you know, what you need to know, and what you have available to you.


Thank you so much for your insight. As a masters student I'm finding that a lot of work is "trial and error" ...
My target protein is part of a two-component regulatory system, so I was thinking of using the kinase that proposedly phosphorylates it in vivo but, seeing as it's a membrane bound protein, wouldn't co-expression be somewhat less efficient if I express them in E.coli BL21 cells? Also, with point mutations, seeing as my protein is unstable, wouldn't this additional negative charge cause it to precipitate more easily?




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