Hi all,
As a negative control, I've used a set-up in which:
1) beads without antibody binding were incubated with the sample and then put on WB. I don't see any heavy chain (which is logic), however do detect my protein on WB?
2) beads incubated with the pre immune sera on WB give a signal for heavy chain (logic, as it contains several IgG's) but also my protein is detected?
Does this mean that in some way, my protein binds to the beads without even needing to couple the antibody? How to solve this?
Hello,
What kind of beads do you use? Some beads come preblocked with BSA and some don't. This could be the issue. Also what kind of buffer do you use to wash the beads? Adding 0.05 % Tween 20 helps too.
Most importantly, changing tubes between washing (and a new one for elution!) helps too as your protein could stick to the surface and you would carry it on the WB. Try changing them event if you use low-bind tubes.
Hope this helps!
If your negative control can detect your target protein, there meaning that more non-specific adsorption. On the one hand, it may be the cause of the beads, such as insufficient blocking; on the other hand, it may be caused by insufficient elution. -Creative Diagnostics
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies