Hi,
i´m looking for a very specific anti-FLAG antibody to couple on magnetic beads. I tried the antibody from Sigma and Pierce, but they seems to be very unspecific in the western blot. Does anybody have a nice tip for me or tried the same?
Thank you very much!!!
Henriette
Hi Henriette,
I am just modifying your protocol a bit.
-blocking : 60 min RT in 5% Skim Milk (PBS).
anti-FLAG 1:2000 in 5% Skim Milk (0.1% Tween PBS) 1h RT.
3x short washing (5 min) 0.1% Tween PBS (4-6 wash X5 min,if you are using ECL prime, from GE)
anti rabbit 1:10,000 in 5% Skim Milk (0.1% Tween PBS) 1h RT. (You can try 1:15,000 to 20,000 dilution)
4 to 6 x 5 min washing 0.1% Tween PBS
I hope, this may help reducing the background.
Another point I am thinking, If your IP wash buffer contains Protease and phosphatase inhibitor that's fine, if not, you can include it in your IP wash buffer, which may protect possible degradation of protein during IP.
GOOD LUCK
Best,
Umakanta
Henriette, have these ABs been affinity purified? I'd make that an absolute prerequisite. In addition, before optimizing the actual AB dilution, have you optimized the detection system (i.e. the secondary AB) regarding dilution? How do you block the beads after attaching the antibodies?
Hi Henriette,
the anti-FLAG M2 antibody from sigma was one of the best antibodies I ever used. I guess your blocking conditions need to be optimized.
Could you attach a picture of the blot?
I usually got very good signals for transfected FLAG-fusion proteins in eukaryotic cells after 1-5min exposure. So if you have to expose much longer you'll probably see unspecific background from secondary antibody. As a control you could simply do a second blot in parallel, without FLAG Ab and only secondary Ab.
Best,
Kai
P.S: Liebe Grüße an den Fritz und euch ein gesundes neues Jahr
Hi Henriette,
The Sigma anti-FLAG M2 ( cat#F1804) is one of the best antibodies. I agreed with Kai's suggestion. You can dilute primary and secondary antibodies more than the standard dilution and give more wash after primary and secondary antibodies incubation if background in Western is an issue.
Best,
Umakanta
Hey Kai and Umakanta,
thank you for answering. Maybe you are right....here is my protocoll and a "nice" picture :-)
-blocking : 10-30 min RT in 5% Skim Milk (0.1% Tween PBS)
anti-FLAG 1:1000 in 5% Skim Milk (0.1% Tween PBS) 1h RT / over night 4°C
3x short washing (2-5 min) 0.1% Tween PBS
anti rabbit 1:5000 in 5% Skim Milk (0.1% Tween PBS) 1h RT / over night 4°C
3x 10 min washing 0.1% Tween PBS
I think the secondary antibody is fine, because I also take it for blots with anti-HA antibodies and they look very nice-means only one single band-where I want it to see.
All the best
P.S. @ KAi: ich werds ausrichten, euch auch einen guten Start
Hi Henriette,
I am just modifying your protocol a bit.
-blocking : 60 min RT in 5% Skim Milk (PBS).
anti-FLAG 1:2000 in 5% Skim Milk (0.1% Tween PBS) 1h RT.
3x short washing (5 min) 0.1% Tween PBS (4-6 wash X5 min,if you are using ECL prime, from GE)
anti rabbit 1:10,000 in 5% Skim Milk (0.1% Tween PBS) 1h RT. (You can try 1:15,000 to 20,000 dilution)
4 to 6 x 5 min washing 0.1% Tween PBS
I hope, this may help reducing the background.
Another point I am thinking, If your IP wash buffer contains Protease and phosphatase inhibitor that's fine, if not, you can include it in your IP wash buffer, which may protect possible degradation of protein during IP.
GOOD LUCK
Best,
Umakanta
The Sigma anti-FLAG M2 ( cat#F1804) is really great - both for Westerns, immunocytochemistry and CoIP experiments. With F2555 rabbit I had problems for immunocytochemistry still worked but not very effitiently
Hi.
I am currently using anti flag sigma cat no:f1804 as my primary antibody and anti mouse for my secondary. However i failed to get any band. As far as i know, this antibody is mouse derived. Any particular reasons why it works with secondary anti rabbit?
Riya, as you are writing, Sigma states that this AB is a mouse monoclonal. The most likely cause that you get a signal with your rabbit secondary is that it is either wrongly labelled.
Your anti mouse secondary also might be wrongly labelled or it has a problem.
You could check any secondary for degradation by SDS-PAGE (sharp bands? smear?) and also if the reporter activity is working (e.g. for ECL, mix 2x100 ul reagents in an eppi and, in the dark room, add 0.5ul or so of your secondary. You should see an immediate bright glow with bare eyes).
Hi Wolfgang,
Thanks for your response. I have not tried anti rabbit for the secondary. I only tried using anti mouse abd failed. However, the previous thread (above) mentioned on their optimized protocol using anti rabbit.
I read from sigma's website mentioning on the species reactivity. It stated "all". Is this why, the antibody might work with anti rabbit?
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