antibody problems in western - Got similiar western signals for three different p (Jul/29/2005 )

for three different primary antibodies, I got almost exactly same signales
I'm thinking maybe the secondary antibody(goat anti-mouse, from santa cruz) is the trouble maker,all the bands appeared are actually non-specific bands, it's not recongizing the real primary antibody at all.

The background is very clear, so blocking should be of no problem.

help me please. thanks a lot~~

-Cathleen-

Could you skip primary antibody, i.e. after blocking step use secondary antibody directly? This will let you know whether your secondary antibody is the trouble maker.

-bullfrog-

QUOTE (Cathleen @ Jul 29 2005, 01:37 PM)

I had exactly the same problem as you have. That bands were caused by your secondary antibodies. Maybe you can try increasing concentration of your primary antibody and see it works?

Good luck!

-Kevin_1999-

Buy a new secondary. Amersham sells secondaries for western of superior quality.

-MaximinaNYC-

Thanks, I did that with my old membranes, stripped them, I still see sth, but the signal is much weaker, I guess the stripping was not complete, so I'm running new gels now.

QUOTE (Cathleen @ Jul 29 2005, 02:37 PM)

-Cathleen-

Thanks, the secondary goat anti-mouse is from Santa cruz, if I prove that's the trouble maker, I'll never ever use their products again, and I'll spread out the word

QUOTE (bullfrog @ Jul 30 2005, 01:06 AM)

Could you skip primary antibody, i.e. after blocking step use secondary antibody directly? This will let you know whether your secondary antibody is the trouble maker.

-Cathleen-

thanks, I did. any recommendations for good primary antibodies? I'm so scared by poor quality antibodies

QUOTE (Kevin_1999 @ Aug 1 2005, 04:26 PM)

QUOTE (Cathleen @ Jul 29 2005, 01:37 PM)

I had exactly the same problem as you have. That bands were caused by your secondary antibodies. Maybe you can try increasing concentration of your primary antibody and see it works?

Good luck!

-Cathleen-

Hi Cathleen,
If you call us immediately when you suspect there is a problem, we can help! Many times the secondary needs to be optimized. The suggestion in this forum of a secondary control is a great starting point. You can optimize the secondary by using different dilutions until your secondary control is clean. Also, if you are using a monoclonal antibody, you need to make sure that the secondary antibody is able to detect that particular isotype (for example mouse IgG2a). These are all things to consider. If you still determine that this secondary is the troublemaker, we'll be happy to issue you a refund.
We can be reached at 1-800-457-3801 ext. 2 or scbt@scbt.com.

QUOTE (Cathleen @ Aug 3 2005, 08:37 AM)

Thanks, the secondary goat anti-mouse is from Santa cruz, if I prove that's the trouble maker, I'll never ever use their products again, and I'll spread out the word

QUOTE (bullfrog @ Jul 30 2005, 01:06 AM)

Could you skip primary antibody, i.e. after blocking step use secondary antibody directly? This will let you know whether your secondary antibody is the trouble maker.

-Immuno06-

hi,
in general upstate and abcam is doing fine in my hands.

gud luck
payeli

[quote name='Cathleen' date='Aug 3 2005, 05:38 PM' post='21085']
thanks, I did. any recommendations for good primary antibodies? I'm so scared by poor quality antibodies

-payeli-