Reuse WB - (Apr/17/2008 )
Hi there,
Yesterday I was developing a WB. The positive control turned out nicely on the Film. The next day I was told, I used the wrong "insensitive" Film and that it might be worth rechecking with a more sensitive film for a longer exposure.
I applied the chemoluminescent dye on my blots, but could not see anything. even the positive control is gone?
So my question:
Do I have to probe with the secondary AB again?
Do I have to probe with both - first and secondary AB - again to see a signal?
What happend to my antibodies?
Thanks
You'd better incubate with first AB and then with the second as usually.I don't think that your first AB's from the first incubation are still alive 
Yesterday I was developing a WB. The positive control turned out nicely on the Film. The next day I was told, I used the wrong "insensitive" Film and that it might be worth rechecking with a more sensitive film for a longer exposure.
I applied the chemoluminescent dye on my blots, but could not see anything. even the positive control is gone?
So my question:
Do I have to probe with the secondary AB again?
Do I have to probe with both - first and secondary AB - again to see a signal?
What happend to my antibodies?
Thanks
if you have a positive result, why to check again???
if you use peroxidase-labeled secondary Ab, H2O2 is substrate but also inhibitor; try to incubate again with fresh secondary Ab
Okay. I only applied a few wash steps and than used the secondary AB. I needed to redo the western, because I used the wrong film, which not sensitive enough. It only showed the positive control.
Everything worked perfect. Thanks to all