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:

enter image description here

enter image description here


1 Answer 1


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.

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .