Reducing background when staining with 1st and 2nd antibodies

Robin Barclay robin.barclay at
Tue Nov 1 19:41:02 EST 2005

Many "anti-mouse" antibodies sold commercially are cross reactive with other species Ig.  Make sure it is affinity purified and cross adsorbed.  Use a Fab2 fragment, if possible, to avoid Fc binding. I heard that goat complete antibodies react minimally with human Fc if you can't get a Fab2, and you can always add in some goat serum before you add your secondary conjugate (pre-add same serum as species antibody was raised in, not human serum). You don't necessarily need IgM-specific - I use an anti-Ig that will bind both IgM and IgG mouse monoclonal primaries. I had to trawl around but came up with an antibody from BioSource that met all criteria and has no non-specific binding if used without primary (my control) - the HRP conjugate of the same antibody is great for immunoblotting.  Of course, you must wash primary and secondary away completely if subsequently using other direct labelling conjugates.
Robin Barclay
  ----- Original Message ----- 
  From: Alan_Stall at 
  To: Cytometry Mailing List 
  Sent: Monday, October 31, 2005 7:30 PM
  Subject: Re: Reducing background when staining with 1st and 2nd antibodies

  If the anti-Mouse IgM is a polyclonal Ab it may have some cross-reactivity to human IgM.	You could do your staining in the presence of human serum.  (there is no perfect Fc block for human, this might also be your best Fc block)  In addition you could use a monoclonal anti-mouse kappa as the secondary reagent.  They tend to show little staining of human cells. 

  Alan Stall 
  BD Biosciences 

       "Elkabetz, Yechiel/Sloan-Kettering Institute" <elkabety at> 
	10/28/05 01:32 PM 
		To:	   Cytometry Mailing List <cytometry at> 
		cc:	   (bcc: Alan Stall/SDCA/BDX) 
		Subject:	Reducing background when staining with 1st and 2nd antibodies 


  I am staining human cells with a mouse IgM as 1st antibody and then
  FITC-anti-mouse IgM as a 2nd. What can I do to reduce background? This
  is because I get a non specific staining of the secondary, which makes
  the negative population shifted to be very close to the positive. Maybe
  an FC block?



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