[Histonet] custom-made polyclonal antibody doesn't stain

[sgoebel <@t> mirnarx.com]

I have done this (or tried to do this) as well and almost ended up bald
from ripping out my hair!!  My first suggestion would be to try to get
down to a 1/25 or 1/50 dilution.  If the background starts to get too
high then try blocking the Fc region.  You can buy commercially
available Fc blockers.  I also tried conjugating the antibody with HRP
which seemed to help some as well.
Good Luck!!

Sarah Goebel, BA, HT(ASCP)
Histotechnologist
Mirna Therapeutics
2150 Woodward Street
Suite 100
Austin, Texas  78744
(512)901-0900 ext. 6912




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Barbara
Verstraeten
Sent: Tuesday, January 04, 2011 8:14 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] custom-made polyclonal antibody doesn't stain

Dear all,

 

I had two antibodies made (in rabbit) for the same peptide.

It was the first time I did this : the firm designed the immunization
peptide, injected and bleed the rabbits and purified the antibody.

I have tested it on paraffin sections: all background, no staining where
it
should be. I tried different retriever buffers and detection with dab
and
fluorescence.

I have tested it on western blot: a lot of background, dirty lanes. To
get a
clearer view I performed a co-IP. Still not really great.

 

Can anybody help me on this issue?

I just want it to work for staining. 

 

Thanks a lot in advance!

 

Barbara Verstraeten, Drs.

Evolutionary Developmental Biology

Department of Biology

Ghent University

K.L. Ledeganckstraat 35

B-9000 Ghent

Belgium

tel: ++32/(0)9 264 52 31

fax: ++32/(0)9 264 53 44

http://www.evodevo.ugent.be

 

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