Hi All
I am recently doing WB fron transfected cells HEK293(using a plasmid tagged by Myc & DDK) For protein extraction i used RIP buffer and protease inhibitor.The Myc antibody i sued from Origene (CAT#: TA150014). After probing the blot with the tag i ended up with high background and multiple bands that exist even in the negative control . The protein concentration i started with was 15 mcg and the tag dilution was 1:1000 . I am kind of new to this area and i am reaching out for help.
Thank You
Hi there,
1:1000 is usual for a primary antibody. What about the type and dilution of the secondary? If you get high background, check the dilution of the secondary antibody and also try to optimize the initial blocking step (using BSA and non fat milk), washes in PBS or TBS containing Tween and the probing steps ( same buffer as washes plus non fat milk).
Hi,
Most Myc tag antibodies I have used so far have the same problem on a western. I partially fixed this issue by introducing an overnight blocking step using 5% nonfat dry milk prepared in 1X TBST. I would encourage you to try this approach, and follow this with three consecutive 1X TBST washes (5 mins each) before adding the secondary antibody the next day.
Good luck!
Anoop
High background noise means you have to improve your blocking step, as suggested above. 5% of BSA or skimmed milk in TBST/PBST for 1hr or more should work fine.
I'm also facing the same problem, I will try once Anoop's suggestion for improved results
Hi Kirankumar Nalla, Did my suggestion help resolve your issue?
Anoop
Hi Anoop Arunagiri, No sir your suggestion did not work for me, don't know where is mistake happening, now planning to change the membrane, previously I used a PVDF membrane, now checking with nitrocellulose membrane
- Badges
- Science topic