Author Topic: Non-specific binding of polyclonal antibodies  (Read 9155 times)

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Offline bmedsci_jude

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Non-specific binding of polyclonal antibodies
« on: May 13, 2006, 08:12:22 AM »
Hi everyone. I was wondering if anyone could help me with some advice? I am a student only a few months old to the field of immunos trying hard to learn as much as I can about immunos to make my staining work for my research degree, staining with a goat polyclonal antibody for a transcription factor that translocates to the nucleus.

I have had some progress since I introduced blocking endogenous avidin and biotin as well as endogenous peroxidase to my procedure in that my negatives are truly negative and there is positive alongside negative staining on the antibody slides, with a little bit of background, giving a confusing picture.

I used antibody dilutions of 1:50 and 1:100 diluted in 1% BSA in PBS. My antigen retrievals were trypsin and trisodium citrate pH6 in HIER. In addition I used BSA, Triton X-100, Tween 20 and PBS alongside my serum in serum blocking, I used a 15 minute soak in methanol to permeabilise my membranes and I used Tween PBS washes after the primary antibody incubation of 2 hours. I also blocked peroxidase at 3% in PBS.  Has anyone any experience with any of these procedures or any advice as to reduce the background and make the resultant picture a little less confusing? I am going to be running an IgG run on the tissue samples Ive been given next week too. Thank you :)
udith Ritchie

Non-specific binding of polyclonal antibodies
« on: May 13, 2006, 08:12:22 AM »

Offline richard03

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Re: Non-specific binding of polyclonal antibodies
« Reply #1 on: November 24, 2008, 12:20:37 AM »
Try different antigen retrieval methods such as Tris-EDTA pH 9. You already used Triton X-100 for membrane permeabilization so there is no need to use methanol. Try to keep your protocol simple!

Offline bmedsci_jude

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Re: Non-specific binding of polyclonal antibodies
« Reply #2 on: November 24, 2008, 03:40:03 AM »
The simplest of protocols didnt work, things got added as I problem solved my methodology and the tissue type I was using (breast). In the end I blocked for avidin, biotin and peroxidase. Took out the Triton X as you said, the methanol permeabilsed enough, I fear the Triton X was destroying the epitopes on top of the MeOH. Used 2% milk in PBS, worked a treat. The AR was eventually trypsin at pH 7.4 at 37oC as per the IHCWorld protocol, worked a treat! Didnt need the HIER on top of that. That was 2 years ago I did that study!! But thanks for your reply
udith Ritchie

Offline richard03

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Re: Non-specific binding of polyclonal antibodies
« Reply #3 on: November 24, 2008, 08:00:47 AM »
Glad you solved the problems!

Re: Non-specific binding of polyclonal antibodies
« Reply #3 on: November 24, 2008, 08:00:47 AM »