I am trying to do Western Blot for PNPLA3. I am using Roti Block as Blocking Buffer and the dilution rate for primary and secondary antibody is 1:10000. I used both ready made gel and handmade 12.5% gel.
However the bands are almost non-existent.
Any suggestion or remark will be of a great help for me. Thank you in advance :)
1. Try increasing the primary antibody concentration.
2. If you are not already doing so, use enhanced chemiluminescence (ECL) detection for maximal sensitivity.
3. If you are using HRP-conjugated secondary antibody, get a new hydrogen peroxide supply, since this reagent is unstable.
4. Try to get a positive control for your blot, which would be purified antigen, or at least a sample known to contain the antigen in detectable quantity.
5. Do not try to reuse blots by stripping them and re-probing. Use a fresh blot for each experiment. To try different antibody concentrations, you can divide a single blot into strips for probing separately.
6. Make sure your secondary antibody-enzyme conjugate has been stored correctly and is not expired.
I would try using the primary antibody at 1:1000-1:2000, hope this helps!
- Badges
- Science method