transfer for 25 minutes, blocking for 3hrs at 37C with 5% BSA in TBS, Primary antibody overnight. Secondary antibody for 1.5hrs. then Exposure with ECL biorad
Hi Shifa
It is always useful to add the Magic Marker from Invitrogen to your Westerns, as this helps identify where the problem might be coming from.
For starters I think transfer for 25 minutes if using semi-dry and 10V is way too short. Are you using wet, semidry or turbo transfer? Also, blocking for 3 hours is too long, reduce this to just one hour and instead of 5% BSA you can just use 1%. If you overblock your western you may lose binding capacity of your primary antibody if it is not great. And please do this at room temperature in a shaking platform and not at 37 degrees. The rest of your paramenters are standard, but please try these tips next time.
Did your coloured protein ladder transfer successfully?
yes ladder came out great..
I am using semi dry apparatus of biorad
one thing more can western blot procedure be completed in one day.. since this take almost two days.
- Badges
- Science method