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trouble detecting phospho-proteins


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#1 holger

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Posted 17 June 2011 - 04:51 AM

Hi guys,

I'm trying to get signals for pAkt and pErk, but have been unlucky so far. The total proteins show up nicely.

As a lysis buffer I use 1% Triton-X in 20mM Tris, 150mM NaCl plus a protease inhibitor mix and Na3VO4. I tried two different approaches; first as I check different concentrations at various time points for a lot of cancer cell lines I thought I could pellet the cells and shock freeze them and lyse them in batches. As I didn't get any signal for the phospho-proteins I lysed the cells in the dish but still didn't succeed. For detection I use the Licor fluorescence system (can detect pAkt + tAkt at the same time).


I'm currently at a loss what to do now. I can use NP-40 instead of Triton but that shouldn't matter, should it?

It would be great if anyone has any suggestions!

Thanks a lot!

Holger

#2 GNANA

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Posted 17 June 2011 - 06:37 AM

If the problem is only in the phosphatase inhibitor, then i would say the use of Na3vo4 a typical tyrosine phosphatase inhibitor might not be sufficient to protect the serine phosphorylation as well, if i am right the phosphorylation is on the serine residue in Akt, so you got to use some more phosphatase inhibitors like sodium fluoride and sodium pyrophosphate as well along with Na3vo4. PMSF and EDTA also would help...

Good luck...

I always had an alternate hypothesis....


#3 protolder

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Posted 17 June 2011 - 02:21 PM

[quote name='holger' timestamp='1308315115' post='112792']
Hi guys,

I'm trying to get signals for pAkt and pErk, but have been unlucky so far. The total proteins show up nicely.

As a lysis buffer I use 1% Triton-X in 20mM Tris, 150mM NaCl plus a protease inhibitor mix and Na3VO4. I tried two different approaches; first as I check different concentrations at various time points for a lot of cancer cell lines I thought I could pellet the cells and shock freeze them and lyse them in batches. As I didn't get any signal for the phospho-proteins I lysed the cells in the dish but still didn't succeed. For detection I use the Licor fluorescence system (can detect pAkt + tAkt at the same time).


I'm currently at a loss what to do now. I can use NP-40 instead of Triton but that shouldn't matter, should it?

It would be great if anyone has any suggestions!

Thanks a lot!

Holger


Hola, I´working now in this area but looking phosphoproteins in tumors after drugs treatments. People in the lab is expert on it, and after not very clears results in our first WB, we´ll do next ones with the following suggestions. They load about 50ug of protein /well. the support for WB will be nitrocellulose better than PVDF because it could have some fluorescence at 700nm. Blocking agent will be specific odissey or BSA better than milk, and washes will be done with TBS/tween better than PBS/tween. As phosphoprotein signals are ligther than non phosphorilated protein, made first this WB of phosphoproteins better than total protein . once you see P-protein made without stripping the total protein (phospho and dephosphorilated).One more thing they work with commercial cocktails of proteases and phosphatases inhibitors.I hope that this could help you, I´ll try again next week. It´s a pleasure contact with somebody which works in the same field.Buena suerte

Edited by protolder, 17 June 2011 - 02:24 PM.





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