Seperating forms of Rb on SDS-PAGE? - should be easy!!!!!!????? (Mar/07/2008 )

Hi,
I have been trying to determine if my protein samples have either or the hypophosphorylated or hyperphosphorylated form of Rb. I have tried several different approaches using different percentages of resolving gel (6% and 8%) and for different times (4-5 hours) at ~25mA. I have tried different monoclonal antibodies from different companies and cannot get anything close to a decent result. Am very DESPERATE for alternatives or any suggestions because this seems like it should be easy but is simply driving me crazy. PLEASE HELP!!!!

Sincerely,
VERY GRATEFUL

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you could try running them on urea page. charge separations are enhanced so you will see the effect of degree of phosphorylation on migration.

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Thanks! Will look into it, any other suggestions would be greatly appreciated!
Do you have a protocol for pouring a urea gel? Or is it simply adding a conc. of urea???

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here it is (it is in word format, the reference is incomplete and probably not entirely correct, anyway), you can prepare other concentrations as you see fit:

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thank you so much. I will let you know how it goes.

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Why the increase in acrylamide/ bis acrylamide? How do you get 60%

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the stock needs to be higher concentration so that there is room for the urea.

we just take 60 gm acrylamide and add 1.6 gm bisacrylamide and bring it to 100 ml (it will go into solution, i promise).

i know that this is really 61.6% but i followed the standard practice of the lab when preparing the stocks (and it worked okay so why change).

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