It seems to be a broad question, but I am trying to detect NOS2 protein after LPS stimulation. I used following conditions but unable to get NOS2 bands. Please suggest if I need to change my protocol:
1. Protein concentration used/sample: 10-12 ug
2. Transfer conditions: Slow transfer (35 volt/overnight, 4 degree) (Transfer buffer: 20% methanol, without SDS)
(After ponceau, very faint bands for high mol wt proteins observed everytime)
3. Blocking: 3% BSA, 90 minutes, RT, shaking
4. Primary antibody: 1:1000,overnight; secondary antibody: 1:10,000 (1HR, RT)
5. Detection method: Chemiluminiscence: HRP substrate
After all this, i get beta actin bands, but not NOS2. Its tricky to find out which step I need to optimize, please suggest if you are aware of NOS2 blotting tips?
Thanks
Your antibody concentrations are pretty low so that may be an issue. Also if you are running the western blot overnight then perhaps you are transferring for too long and you have pushed too many of the proteins through the gel?
Thanks for the answer, is it not good to transfer slowly and overnight to ensure efficient transfer of large proteins? What if my protein of interest is not transferred then even if I optimize other steps, it won't give me correct band. Please suggest.
We transfer at 30-40 mAmp for about 1.5 hours. We watch the the sample line migrate out of wells and stop it right before it runs off the end of the gel
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