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I have been working on optimizing western blotting for Caspr (contactin associated protein), but I am having difficulties and would like suggestions.

  1. I ran my proteins on a 40% acrylamide gel,
  2. I used TBST for washing and BSA as blocking buffer.
  3. I incubated the blot with primary antibody overnight.
  4. The secondary antibody is linked with HRP.

I could not find any bands in the blot.(Same problem was faced in case of contactin) What might be the reason?

Edit:-

  • I repeated the experiment a couple of times and found that secondary antibody(Anti-mouse,1:5000)wasn't working anymore.

  • I made freshly diluted secondary antibody again and found bands.

  • The old secondary antibody dilution was only few months old.What may have gone wrong with it?

blot images

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  • $\begingroup$ What percentage is your gel? And: Did you do Ponceau staining of the membrane? $\endgroup$ – Chris Jun 14 at 14:34
  • $\begingroup$ The resolving gel was 8%. Yes, Ponceau staining was done. $\endgroup$ – Lekshmy.R Jun 14 at 14:35
  • $\begingroup$ And there you did see bands I assume. How old is your antibody dilution? $\endgroup$ – Chris Jun 14 at 15:31
  • $\begingroup$ Do you have a positive control (actin or something)? $\endgroup$ – WYSIWYG Jun 15 at 16:40
  • $\begingroup$ Usually you visualize the bands on the blot by exposing the blot to X-ray film or otherwise visualizing the HRP reaction, because you can't just see the reaction on the blot. Can you show us a film or other exposure of the blot? The blot itself looks ok but it's hard to say what the problem is without seeing such an exposure. $\endgroup$ – Maximilian Press Jun 16 at 16:53

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