I had been using "poly rabbit anti-iNOS/NOS Type II" from BD but i am not really happy. For fluo-IHC it works but not that good (reproducible) and with western...no signal at all. I would be happy to swithch to another company or if someone has any additional tricks for this one, i am happy to hear them.
Thanks a lot for the info. (actually this was the next one on my list to order)
Hi, we had iNOS/NOS Type II (610333) from BD Transduction Laboratories for western blot and it worked well ... did you try a positive control like proposed by BD (mouse macrophage lysate/RAW cells)?
Hi, thanks for the answer. This would be the antibody (610332) that i had used also. I did not try positive control (due to lack of cultured macrophages). In the model that I am working on pathology is driven by large quantities of infiltrating macrophages (as seen also with positive iNOS immunofluorescence). I was hoping to quantify that using WB on tissue lysates but unfortunatley I had managed to get a positive result (very nice/clear signal in inflammed tissue and no signal in controls) only once that I could not reproduce using same experimental setup (even same tissue lysate). Would it be possible to get info on your protocol (incubation time/buffer, Ab concetration)?
Hi, you can buy the positive control Mouse Macrophage + IFNγ/LPS Lysat (Cat.No.: 611473) ... normally we block the membrane using fat free milk, Ab incubation 1:1000 1 hour at room temperature or +4°C over night. No special protocoll. We tested also another iNOS-Ab which also worked fine http://www.rndsystems.com/Products/MAB9502.
May be you have no positive band because you "dilute" your macrophage protein with other tissue proteins preparing the tissue lysate? I would recommend to perform iNOS fluorescence immunohistochemistry and quantify the brightness ... or count the iNOS positive macrophages in relation to the total number of macrophages stained with e.g. CD68.
You seem to have received good advice already regarding for your BD sourced antibody, which hopefully works for you. We have also used iNOS antibody from Santa Cruz previously, similar to your first answer.
Does anybody know how much Antibody (Abcam ab3523 and SantaCruz SC-650) in which dilution is preferred for Western Blot of Rat tissues (Liver, Kidney, Brain and spine)?
Aleksandar how did you used to prepare your tissue? I'm having similar problems and I think it's the way I prepare
- Badges
- Science topic