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serum and Akt phosphorylation - Do I have use serum-free media to check pAkt when western blotting? (Jun/04/2008 )

Hi all,

I plan to compare the levels of activated Akt (pAkt) by western blottinig between Pten wild type and Pten knock-down cells to see if Pten regulates Akt activation or not.

I wonder if I have to use serum/growth factor free media to check accurate phosphorylation levels between two groups.

In my experience, I used to use serum free media to check Erk phosphorylation because serum can induce Erk phosphorylation dramatically, but I do not know about Akt.

Also, how long do I have to do serum-starvation if I have to use serum free media?

Please Pten/Akt researchers give me some advice!!!

Thanks!!

-cancerous-

QUOTE (cancerous @ Jun 4 2008, 12:18 PM)
Hi all,

I plan to compare the levels of activated Akt (pAkt) by western blottinig between Pten wild type and Pten knock-down cells to see if Pten regulates Akt activation or not.

I wonder if I have to use serum/growth factor free media to check accurate phosphorylation levels between two groups.

In my experience, I used to use serum free media to check Erk phosphorylation because serum can induce Erk phosphorylation dramatically, but I do not know about Akt.

Also, how long do I have to do serum-starvation if I have to use serum free media?

Please Pten/Akt researchers give me some advice!!!

Thanks!!


You may (perhaps) find answer in one of these links:
http://search.vadlo.com/b/q?sn=158621799&a...serum&rel=0
..

-cellcounter-

QUOTE (cancerous @ Jun 4 2008, 01:18 PM)
Hi all,

I plan to compare the levels of activated Akt (pAkt) by western blottinig between Pten wild type and Pten knock-down cells to see if Pten regulates Akt activation or not.

I wonder if I have to use serum/growth factor free media to check accurate phosphorylation levels between two groups.

In my experience, I used to use serum free media to check Erk phosphorylation because serum can induce Erk phosphorylation dramatically, but I do not know about Akt.

Also, how long do I have to do serum-starvation if I have to use serum free media?

Please Pten/Akt researchers give me some advice!!!

Thanks!!


serum starvation is a double-edged thing: it may cause stress and may activate apoptosis; pro-survival function may involve phosphorylation of Akt

dependence of Akt on PTEN is not novel but I think you have a novel regulatory mechanism under examination...

-The Bearer-

QUOTE (cancerous @ Jun 4 2008, 09:18 PM)
Hi all,

I plan to compare the levels of activated Akt (pAkt) by western blottinig between Pten wild type and Pten knock-down cells to see if Pten regulates Akt activation or not.

I wonder if I have to use serum/growth factor free media to check accurate phosphorylation levels between two groups.

In my experience, I used to use serum free media to check Erk phosphorylation because serum can induce Erk phosphorylation dramatically, but I do not know about Akt.

Also, how long do I have to do serum-starvation if I have to use serum free media?

Please Pten/Akt researchers give me some advice!!!

Thanks!!


Hi,
May I ask you about your experience about pERK? I am going to do western for pERK on a cell line to check if the drug is inhibiting ERk phosphorylation or not. I plate the cells, next day

starve them with serum-free medium 0.1% BSA, and next day treat them for 5 minutes, then lyse the cells. What I can't decide when I'm treating the cells 1. shall I remove the starvation

medium and prepare a new solution with drug and starvation medium and treat the cells? or 2. shall I remove the starvation medium and prepare a new solution with drug and 10%FBS

and treat the cells) or 2. add the treatment on top of starvation medium? . I will be grateful if you can help me, Thanks,

-enthusiastic-

Probably cell line dependent. In my experience, growing (non-starved) HEK 293 cells do not give pAkt signal on a western blot. Pten kd cells should give you increased pAkt so it will probably give a signal with or without starvation. Normally I starved overnight, but this again can be cell dependent. In some cells apparently 3-6 hours starvation is optimal and longer can lead to stress response.

pERK can be detected with/without starvation.

-Michelle4-