Reproducibility - (Jan/18/2007 )
Hi Guys
Last week I did a time response experiment to study the effect of a growth factor on erk pathway. I treated the cells for 5, 10, 15, 30, 60 minutes and lysed them n them ran western n probed for phospho erk 1/2. i ontained a very nice time response for a duplicate experiment ( two times done at the same time). I picked the point (15 minutes) at which maximum activation occured and wanted to study the effect of drugs on this activation however i coud not obtain the activation again ( I could nto see the response i saw last week). I dont know why ??????????????????????????????
Ill appreciate any help,,,,,,, thanks
Last week I did a time response experiment to study the effect of a growth factor on erk pathway. I treated the cells for 5, 10, 15, 30, 60 minutes and lysed them n them ran western n probed for phospho erk 1/2. i ontained a very nice time response for a duplicate experiment ( two times done at the same time). I picked the point (15 minutes) at which maximum activation occured and wanted to study the effect of drugs on this activation however i coud not obtain the activation again ( I could nto see the response i saw last week). I dont know why ??????????????????????????????
Ill appreciate any help,,,,,,, thanks
I recapitulate to check if I´m right: you did ERK phosphorylation kinetics with peak maximum phosphorylation after 15 min of induction; then you did kinetics after drug treatment but ERK was not phosphorylated;
or you did the same experiment two times with totally different results?? (if the last assumption is right, try a third and fourth time)
to give clues it might be useful to tell us more about the drug or, at least, kind of drug
I did the first experiment that showed maximum activation of erk after 15 min treatment. IT was in duplicate and I got the same exact results for both of them. when i tried the same exact experiment the 3rd time., I did not get any erk phosphorylation.. I dont know why till now, is it something wrong with the cells or the growth factor i'm using ??????
Last week I did a time response experiment to study the effect of a growth factor on erk pathway. I treated the cells for 5, 10, 15, 30, 60 minutes and lysed them n them ran western n probed for phospho erk 1/2. i ontained a very nice time response for a duplicate experiment ( two times done at the same time). I picked the point (15 minutes) at which maximum activation occured and wanted to study the effect of drugs on this activation however i coud not obtain the activation again ( I could nto see the response i saw last week). I dont know why ??????????????????????????????
Ill appreciate any help,,,,,,, thanks
I recapitulate to check if I´m right: you did ERK phosphorylation kinetics with peak maximum phosphorylation after 15 min of induction; then you did kinetics after drug treatment but ERK was not phosphorylated;
or you did the same experiment two times with totally different results?? (if the last assumption is right, try a third and fourth time)
to give clues it might be useful to tell us more about the drug or, at least, kind of drug
are you sure that your kinase is still active? it may have denatured in the time between assays.
Last week I did a time response experiment to study the effect of a growth factor on erk pathway. I treated the cells for 5, 10, 15, 30, 60 minutes and lysed them n them ran western n probed for phospho erk 1/2. i ontained a very nice time response for a duplicate experiment ( two times done at the same time). I picked the point (15 minutes) at which maximum activation occured and wanted to study the effect of drugs on this activation however i coud not obtain the activation again ( I could nto see the response i saw last week). I dont know why ??????????????????????????????
Ill appreciate any help,,,,,,, thanks
I recapitulate to check if I´m right: you did ERK phosphorylation kinetics with peak maximum phosphorylation after 15 min of induction; then you did kinetics after drug treatment but ERK was not phosphorylated;
or you did the same experiment two times with totally different results?? (if the last assumption is right, try a third and fourth time)
to give clues it might be useful to tell us more about the drug or, at least, kind of drug
if your experiment subsequently two-times succeed there is some likeliness that your third experiment failed by any reason; so, better try a fourth time and see what will happen; that kinases do adaptation kinectics is normal and do no kinetics may only explained by absence, total inhibition or denaturization;