Hello, researcher
I would like to ask about my western blot results
Last week, I performed dot-blot analysis on nitrocellulose membrane using mouse anti his 1:2500 (1st), and goat anti mousre IgG HRP and DAB substrate (sigma). I can detect positive results.
Now, I repeat the dot-blot again but using sing mouse anti his 1:1000 (1st) (new tube, same company), However, i get a negative result,In addition, the positive protein control also showed negative results.
So, i want to know Why?
[My dot-blot protocol 1. dot 2. block in 5% nfdm in TBST 1h 3. probe with mouse antihis 1:1000 over nigth 4 degree celcius 4. probe with goat anti mouse 1:5000 1h, rt 5. Add substrate mixture (DAB 3mg; sigma in 5ml TBS, 25ul of NiCl2 and 4ul of 30% H2O2)]
Thank you so much