Membrane Froze - Did this ruin my results? - Western Blot not working anymore?? (May/05/2008 )
Hi everyone,
I've been working with protein extracts from bloodsamples and western blotting for a particular protein. My first blood set I used, I got really nice reproducible results (Ran 12 samples, each three times....freeze thawed each time and it was fine) However now I've gotten a new bloodsample set, did the protein extraction and actually seemed to get a higher concentration of protein. However now when I run the samples I am getting VERY weak signal using the same antibodies and protocol. I even ordered new antibodies (primary and secondary) incase somebody left them out and they weren't working. While waiting for the new antibodies I left two test blots in the fridge, they were in blocker (5% skim milk in PBST) but weren't agitated and they froze. I got TERRIBLE results. the antibodies stained some weird non specific bands, mostly in the running front (I even used one of the old bloodsamples which had been reproducible).
Was this caused by the freezing?? could this ruin the membrane??
I can't think of what else is going wrong and it's terribly frusterating (ponseau showed a 'normal' blot)
I've been working with protein extracts from blood samples and western blotting for a particular protein. My first blood set I used, I got really nice reproducible results (Ran 12 samples, each three times....freeze thawed each time and it was fine) However now I've gotten a new blood sample set, did the protein extraction and actually seemed to get a higher concentration of protein. However now when I run the samples I am getting VERY weak signal using the same antibodies and protocol. I even ordered new antibodies (primary and secondary) in case somebody left them out and they weren't working. While waiting for the new antibodies I left two test blots in the fridge, they were in blocker (5% skim milk in PBST) but weren't agitated and they froze. I got TERRIBLE results. the antibodies stained some weird non specific bands, mostly in the running front (I even used one of the old bloodsamples which had been reproducible).
Was this caused by the freezing?? could this ruin the membrane??
I can't think of what else is going wrong and it's terribly frustrating (ponceau showed a 'normal' blot)
if it was caused by the freezing then you would expect a generalized or random effect, not bands in the lane or at an interface (unless they were altered and had an epitope that the antibody recognizes exposed).
ice crystals may damage the membrane but, if the transfer had already been performed, and the samples are reasonably stable to freeze-thaw, then you should still have obtained a reasonable result (i think).
As mdfenko I doubt that the freezing was the cause of your problem I store all my membranes dry frozen and never had any trouble. As for the fridge freezing stuff it happens to me once 'cause the blot wa too close to the back of the fridge but I didn't get any bad trouble with the membrane