I encountered some problem to detect TLR3 for my western. I did not get any bands. After reading a bunch of literature, I am not sure exactly what I did was wrong. I appreciate if anyone has worked with TLR3 to give me some tips.
Buffer: RIPA to lyse DLD-1 cells for 30 mins at 4oC
Lysate was cleared by centrifugation but no sonication
65 ug total protein was loaded each well. Lysate was loaded with the LDS non reducing sample buffer
I also tested a range of sample denaturing conditions before loading the gel: boiling at 95oC for 5 min, 65 for 5 min, 30oC for 5 min, 0oC for 5 min
I read that some people said for membrane proteins, do not boil them, but also other papers published beautiful data with their samples boiled at 95oC.
Toll like receptors are membrane proteins, maybe you should try to optimize our isolation results by corresponding isolation Kits (optimized for membrane proteins). Another potential source of uncertainty may be unknown specificity/reliability of the utilized antibodies for detection (e.g. see Baker M. Reproducibility crisis Blame it on the antibodies. Nature. 2015_521.p274-6. doi 10.1038521274a).
I would propose to check and revise the used protocolls for protein isolation and Western blot analyses (for adequate protocolls corresponding literature should be consulted).
Is it the first time that you use this antibody for WB, what is its reference? Have you tried optimizing its concentration etc? A positive control in the gel would be great. At what speed did you do the centrifugation step?
Centrifugation step/speed is crucial here.
65 ug is too high; reduce the amount of loading protein.
Include a positive control which will clarify that your antibody is working or not
If it is working ok, then you can focus on your experimental sample preparation.
- Badges
- Science method