I am checking my target gene expression by northern blot analysis. I got a strong band in all samples which contains my target gene transcript and positive control and no band in negative control. Now, I want to check the GAPDH without scrapping the previous probe (as I need the two band in same film).My target band size is 687 bp and GAPDH transcript size is 1 kb. My previous probe showed strongly exposed bands. I tried to reprobe without scrapping but the probe doesn't bind at all. I am confused whether my gene is present or not. Please suggest me how can I solve this?
Yes, by the word 'scrapping' I meant stripping.First, I probed with my target gene.Now, I want to reprobe with GAPDH without stripping as I want to keep two band (one is my target gene and another band from GAPDH). I tried it before but GAPDH doesn't bind.
Do you use transcript probes or oligos? You can exclude the possibility that your GAPDH probe is not functional by pipetting some of your probe template on a membrane and hybridize it as a positive control. If there is no strong signal you can be sure that your probe design is faulty. On the other hand, if your target signal is very strong it might outshine the GAPDH signal on the same membrane.
Paul Bolay Thank you for your suggestion. I use oligos. I will check the GAPDH the way you mentioned.
Hi, did you figure this out? I also need to do the same thing and am trying to figure out the best way to do this. Are your probes radioactive?
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