I have checked Cg A, Vmat 2 AQP4 from CSF.. and all of them appears at 50KD. I don't know what is going on.
these are non specific bands, you should use different dilutions of primary antibody first then if u are not getting result change the dilution of secondary ab. Do proper washing of blot.
Probably are non specific bands. You should use different solution blocking (BSA or milk) and tthe other option is the secondary antibody
What are the lysates that you are running on your gel? How are you treating them/preparing them?
Looks like you are only detecting immunoglobulin. Dilute more the second antibody, try to load more protein, and add a more stringent washing.
A ±50kD band may result from the heavy chain of an antibody. I am guessing you are performing IP experiments and put this on WB? The antibody that was coupled to the beads will end up in your eluate, and a secondary antibody might detect these (or a ±25kD band, corresponding to the antibody light chain). You can try to use a secondary antibody that cannot recognize the antibody you IPed with:
1) use a isotype specific antibody to detect your primary antibody in the WB, which cannot detect the antibody used for the IP
2) Use antibodies of different species for IP and detection
3) if your desired band runs well above 25kD, use a secondary antibody that is light-chain specific. This way, you won't detect the ±50kD fragment, which is often given problems for detection of many proteins (the 'average' protein size runs at about this height...).
4) Use the clean-blot system. This also appears to work quite well for some.
cheers
Cheers
Hi Leandro, you are probably detecting non-specific bands, you can start by changing the dilution of your secondary antibody, also use washing with TBST and either BSA or powder milk, that one works fine for me. A more suitable option is to use a light chain specific antibody to avoid detection of the heavy chain ~ 50 KDa that always give you a lot of noise. Goodluck. Saluda a Agustin de mi parte, un saludo , Carlos
- Badges
- Science method