reporter gene -seap - expression of seap is very low (Jul/07/2007 )
Fred-33...have u seen any difference in ur seap level with and without P/S in medium??? what FBS (final) conc u had used ??
Today, i have changed my medium after transfection with DMEM +5% FBS with and without P/S.
(( I usually use 10% FBS) but for seap assay and to get low background level, i have reduced it to 5%.))
P/S should not affect SEAP expression. Normally, you should use the same culture medium for maintianing these cells post transfection. Your concern with residual AKP activity in the serum is valid and I think 5% FBS is fine for many cells.
One note: SEAP has long halflife and can accumulate over time during your experiment, therefore it does not serve the purpose as a reporter for promoter strength, because you should see your SEAP activity goes up over time. This may create some confusion under some setting. Reporter with short halflife, such as luciferase does not have this problem.
I've done promoter expression level analysis with SEAP, and results seemed fine to me... so you make me in trouble.
I think like genehunter that 5% should be ok and that P/S doesn't change things too much.
fred_33: www.pnas.org/cgi/content/full/0606398104/DC1
thank you 