Hello,
I'm trying to utilize a native gel for a protein dimer experiment. Everything seemed to work relatively well until I try to examine the blot in a ChemiDoc MP. I don't see any antibody binding at all. The blot is completely empty (slightly cloudy looking).
Running the blot with a BioRad precast 7.5% gel using these conditions
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4781702/pdf/jove-107-53723.pdf
Running buffer has no SDS in it nor does sample buffer.
I transferred the gel with a BioRad turbo blot (1.3A/25V20 mins) constant A. I used the 5x buffer which has ethanol in it. I quickly stained gel with Revert total 700 and noted protein in the 4 lanes I loaded. The ladder transferred as well.
After I incubated O/N with a primary antibody and followed the traditional WB steps for washing and secondary antibody. Any ideas or suggestions on why I saw protein on the blot, however no signal at all when using the antibody?
Hi Jared,
Did you treat the sample with reducing agents like 2-Mercaptoethanol that could affect recognition by the antibody?
If you Ponceau or Amido Black stain the membrane, can you see your expected band ? Alternatievly, can you see the desired band after you coomassie stain the gel after transfer ? If yes, then you need to verify whether the primary antibody is viable. Try to repeat the experiment with an adequate amount of posetive control protein that does interact with the antibody. Also, a fresh lower dilution of the primary antibody might help.
hi
You should check if the antibody is able to detect the native form of the antigen( by Immunoprecipitation (IP) the protein lysate and then check the WB in native & reducing condition)
good luck
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