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Currently using dot blot to attempt to determine if a serum contains antibodies for some reagents I am testing.

  • I pipetted samples as 10-5ul dots on whatman paper.
  • Incubation steps were for 1 hour with light shaking.
  • Blocking solution is composed of 1mg/ml BSA in PBS.
  • Wash steps were 3X PBS for 10 minute each.
  • Solution for incubating the blot in primary antibody was 1ml of serum per 9ml of blocking solution.
  • Solution for incubating the blot in secondary antibody was 100ul of IgG in 10ml of blocking solution.
  • Then a 1:10 DAB substrate mix was used to develop the blot. Products used for the test listed below.

Currently, the whatman paper is a light brown with vague outlines of the dots where the samples were pipetted. Samples that should trigger more antibodies than others (like serum positive control) tend to be a bit darker, but it's not nearly as clear as we'd hope for.

I have also blotted the IgG which usually forms a darker smudge, rather than a clean, dark dot. Does anyone have any advice on how to improve the developing process? I have also tried using TBS, adding Tween to the PBS or TBS as well as trying nitrocellulose instead, but none of these have impacted the results. Reagents:

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In the absence of another answer, I'll transition my comments to an answer.

  1. Please label your images.
  2. Both an Abcam guide and a Thermo troubleshooting document suggest using a kit or DAB protocol including metals such as nickel or cobalt to intensify signal. I notice that the kit spec that you link doesn't seem to have these. Have you tried different DAB kits?
  3. They also recommend carefully monitoring staining over time under a scope and removing as early as possible. This may help to reduce background.
  4. I'd try a few different combinations of conditions, stain + wash times, to see if any have an effect. I don't have a great sense of where your protocol came from, I'd suggest clicking through those documents above or searching the literature to find more DAB protocols that you might try.
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