[Cytometry] preventing non-specific Ab binding in bovine samples

Ruth Nissly rah38 at psu.edu
Mon Mar 12 12:30:50 EDT 2012


Thanks, everyone, for your input about the problem of goat-anti-mouse
secondary binding directly to bovine blood cells.  I received many emails
with a lot of suggestions.  Here is a summary of the input, with 3 possible
causes and methods to correct the issue.  (Side note, I know that she has
tried using bovine serum albumin, but not sure if heat-inactivated serum has
been tried.)

 

I will pass along these suggestions to the client having the problem.  

 

Cause:  Improper processing of sample

*Contact research labs with experience in bovine flow cytometry, including
those at Washington State University, University of Cambridge, University of
Guelph, and Moredun Research Institutes

*Include additional wash step after staining

 

Cause:  FcR binding

*Use 2% gamma-globulin-free horse serum to block non-specific binding

*Stain, wash & fix in the presence of 10% bovine serum

*Use Fab fragment for secondary (suggested multiple times)

 

Cause:  Cross-species reactivity between anti-mouse Ig and bovine Ig (known
issue with Ovine cells)

*Pre-absorb secondary with bovine serum

*Dilute secondary in 1-2% bovine serium

*Try a different secondary/different supplier, particularly one that is
adsorbed

 

General tips

*Directly label the primary antibody

*Purchase directly labeled primary antibody

*Ensure serum used for blocking is heat inactivated

 

Thanks again!

Ruth Nissly

 

Research Technologist

Microscopy & Cytometry Facility

Huck Institutes of the Life Sciences

Penn State University

 

W124A Millennium Science Complex

University Park, PA  16802

 

814-863-2762

 <mailto:rah38 at psu.edu> rah38 at psu.edu

 



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