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Western blot

  1. Nov 29, 2011 #1
    Hi Forum,

    I have been working with Western blot for a while now. I got beautiful results in the beginning when I started the experiment. Now I am stuck with three more antibodies, which I have been trying for the last 5 months, and not getting any result. I could not get any band at all. I do not know what is going wrong. I took help from people in my lab who at least get bands from my own sample. I felt that there was something wrong with my reagents, but when I changed the reagent, even then I could not get results. Please, is there anybody to help me with my situation.

    Thanks
    Selvar123
     
  2. jcsd
  3. Nov 29, 2011 #2

    Ryan_m_b

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    Staff: Mentor

    Hi there Selvar, perhaps if you could outline exactly what reagents, antibodies, protocol etc you are using so that members could get an idea of what is possibly going wrong.
     
  4. Nov 29, 2011 #3

    Monique

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    Staff Emeritus
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    Gold Member

    What are you saying here? That other people do get bands with your sample and antibody, but you don't?
     
  5. Nov 29, 2011 #4
    Hi Forum,

    Thanks for the reply. I am trying to work with dCK, TK1 and PCNA antibodies. I could not get bands for dcK or TK and the band that I got for PCNA was not satisfactory, the PCNA is supposed to be a strong antibody, but it did not show sharp bands. This was the case with me as well as my colleague.

    I use reagents prepared in the laboratory, consisting of tris-HCL buffer (running and transfer buffers), reagents for preparing gels such as 1.5M and 1M tris, 30% acrylamide, sds, 10% fresh APS, TEMED. I prepare 12.5% gel everytime. I load protein according to Bradford assay by plotting the standard curve, so that I have equal concentration of protein in each well.

    Once I load the gel, I run it for 45 minutes and put it for transfer in cold room for 2 hours. I block the membranes with 5% non fat milk for an hour and incubate it overnight with primary antibody. the next morning, I wash the membrane 3 times and incubate with secondary antibody for 1 hour. I would wash the membrane several times and place it in ECL 1:1 diluted. Then, I would take the membrane for filming.

    I DO NOT do the coomassie stain.

    I hope this would help people to help me to troubleshoot. Please suggest if any other information is needed.

    Thanks
     
  6. Nov 29, 2011 #5

    Ygggdrasil

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    How do you know this? Sometimes antibodies that work for one purpose (e.g. immunofluorescence) do not work well for other purposes (like western blotting). Are there antibodies from other suppliers that you can try?
     
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