block for polyclonals

From:Gayle Callis

John bring up some good points.  You can also purchase BSA from Jackson
Immunoresearch that is immunoglobulin and protease free, recommended when
working with large animal antibodies but it works with others also.  It
comes lyophilized, and will not break the bank.    

We frequently make up an inhouse normal serum block and make one portion
with avidin blocker (1 drop per ml, Vector Avidin) then the second portion
with Vector biotin block (1 drop per ml).  Be sure to put the drops in the
tube FIRST, then add you serums, etc and qs with buffer - this way you do
not dilute the serums in Normal Serum block.  Since we block with NSB for
30 min or so, just split the time - half with the avidin/NSB combo and half
with biotin/NSB combo - anything to save time.   Vector has other
recommendation for using biotin with the primary antibody but have never
used that, it will work too. 


At 03:48 PM 2/22/02 -0800, you wrote:
>   Linda, I  didn't respond at first until I re-read your question after
>Patsy's reply. I'm  guessing from what you wrote that your human tissue is
>formalin fixed rather  than the acetone fixation you used for the cell
>pellets. If so I would  definitely retiter the antibody using formalin
>fixed material. However, another  possible problem is that you are adding
>avidin to your blocking serum. This will  tie up any endogenous biotin that
>may be around, but now you have the problem  that you need to neutralize
>any unbound avidin that may be deposited on your  tissue. You can do this
>by adding biotin to your primary antibody. I find  Vector's A/B blocking
>kit an easy way to do this. You might also try switching  to a casine block
>instead of a serum block. Again, I find Zymed's CAS block to  be an easy
>way to do this. Finally, you are using BSA in your block and perhaps  in
>other buffers. Make sure you are using a high quality BSA. Cheaper, less 
>pure, BSAs can contain proteins that interact with IgGs and lead to
>nonspecific  background. This is especially true for goat primary
>antibodies. I spend several  days trying to titer a goat polyclonal ab and
>could not get rid of the  background stain until I realized that the BSA in
>my buffer was reacting with  the anti-goat secondary. When I changed to a
>high purity molecular biology grade  BSA my problem went away. Similar
>background problems can be seen with some  casine blocks that are not
>highly purified, but I have not seen the problem with  CAS Block. Hope some
>of these suggestions help.    John E. Tarpley 5-1-A
>Associate Scientist
>Amgen  Inc.
>One Amgen Center Drive
>Thousand Oaks, CA 91320
>These Opinions are  my own and not necessarily those of my employer.    
>-----Original Message-----
>From: rueggp    []
>Sent: Friday, February 22, 2002 2:39    PM
>To: Linda Hylander
>Subject: Re: block for    polyclonals
>    Another possible fix would be to use an avidin/biotin    block or a
>detection system that doesn't use AB such as DAKO Envision two step   
>labelled polymer system, in case your background is endogenous biotin which
>   may show up in your frozen sections and not in your cell pellets. 
>Patsy    Ruegg    Linda Hylander wrote:                I was wondering if
>anyone has      suggestions to eliminate background when using polyclonal
>Ab. I work with      frozen cell lines and tissues, I rarely do formalin
>fixed      IHC.Problem: I      titered out a primary polyclonal Ab fine
>with no background on a frozen cell      pellet, acetone fixed slides, Ab
>concentration 2.5ug/mL. I stained frozen      tumor breast tissues, had
>loads of background on my isotype control      sections. My secondary Ab is
>goat anti-rabbit, biotinylated, I blocked with      10% goat serum, avidin
>and BSA. I appreciate any ideas and many      thanks.  
>Linda Hylander HT,  IHC(ASCP)
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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