Hello
I tried to use protein A Dynabeads to bind the Fc part of a rabbit antibody that I will use to capture specific exosomes. My problem is that the blank signal is too high and there is not too much difference between blank and spiked samples. I thought to use a blocking buffer to avoid nonspecific bindings but then, I read BSA would not be so efficient because it does not work well with hydrophilic surfaces (which is my case). I tried glycine 0.5 M for 2 hours but no improvement. Any suggestions?
Thank you in advance
hi again,
Blocking with BSA will work best on a hydrophobic surface, where surface adsorption keeps the blocker in position. Dynabeads™ Protein A and Protein G have a hydrophilic surface. Hence, BSA blocking may not be very successful, as surface adsorption is not promoted between the hydrophilic surface and the hydrophobic BSA. Instead, reduce non-specific binding by performing more stringent washing and adding Tween-20® detergent (concentrations of 0.01-0.1 %) to the washing buffer.
- Use a more stringent washing buffer for washing.
- Add a non-ionic detergent (Tween® 20 or Triton® X-100) to the washing buffer, in concentrations between 0.01 - 0.1 %.
- If the beads are blocked before precipitation, add identical blocker to the washing buffer.
- Increase the number of washing steps.
- Prolong the washing steps.
- Decrease incubation time (beads and sample).
- Try the indirect method.
- Decrease the antibody concentration.
- A pre-clearing step may be performed to remove molecules that non-specifically bind to the Protein A/Protein G or the beads themselves.
kind regards
ketil
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