can detet anything with my western membrane - (Oct/30/2007 )
hi, I have problem with replicating my western result. For once, I have detect the protein with antibody from Chemicon, mab3057. Yet, as I repeat the same procedure with cell lysate from same cells, I wouldn't detect any signal. i use PVDF membrane, as blocking and antibody dilution buffer, 5% nonfat-milk dissolved in 0.05%PBST. blocking 1hr at RT, primary antibody 4oC O/N, PBST wash 3X 10min each and detection antibody 1hr, RT. I use ECL from Western Lighten. I've checked the protein on the membrane with Poncrusa solution that it does exist. I would really appreciate for any suggestion!!!! Thank you for all the help!!
You don't mention a secondary antibody. Is your first antibody labelled or have you forgotten to use the second one? This would be an explanation for not detecting anything
You changed nothing in your protocole, and now you can't see anymore your protein?
Do you have any background (maybe your ECL suubstrate is no more good ; you should be careful not to contaminate solution 2 with solution 1 and vice et versa).
get a new aliquot of the primary and secondary antibody. We have had similar problems and the antibodies were the culprit. Make sure that the right secondary antibodies were used.
thank you all for replying and thanx for all the suggestions
my primary antibody is anti-mouse OPN and secondary antibody is anti-mouse HRP...
However, as I redo my experiment with new aliquot of primary and secondary antibody and change a new lot of ECL, the result are the same that no signal was detected!!
Do you have a positive control? I mean, could load load again the cell lysate that gave you a signal previously? (I'm not talking about loading extracts from the same cells, but really loading the same extract)
Are you sure about the transfer? what about doing a Ponceau staining of the membrane? just to be sure that you have the proteins.
Your using a cell lysate from the same cells you said. Did you freeze thaw it, because we've had issues with that. You don't see anything on the western or no proteins?
And indeed, did you have a positive control?
i have checked the protein on the membrane with Ponceau staining and confirmed its presence and as for positive control I load a wt cell line that is sure to express the protein of interest. The strange thing is that even if I used the fresh prepared sample or sample that have been through 1~2 times of thraw freezing cycle, I would not be able to detect it..or have a repeatable result with same working condition!! I have changed all the antibody and buffer to be newly prepared but nothing seems to work!! I would really appreciate any suggestions!!! Thank you all for the help!!