[Histonet] protein A problem with Staph aureus immunostaining

[Jason Palmer]

Hi all, 

  

A question re. immunostaining of bacterial antigens.  We are trying to stain cultured MRSA (Staph. aureus) with an antibody to PBP2a, which is the protein that confers antibiotic resistance to these bugs.  So far we are finding non-specific binding of our antibodies to protein A is a real problem (or at least we assume that protein A is the problem).  Primary is rabbit anti PBP2a and we follow this with a Cy3- conjugated anti rabbit IgG.  We have tried normal serum blocking (10% rabbit) before the primary, as well as an Fc fragment of rabbit IgG as a more specific blocker of protein A before the primary, but so far without much success; we see labe ling with our primary, with an isotype control and also with diluent alone.  So it would seem that both our primary and secondary is binding to protein A non-specifically. 

  

Next things to try are increasing the concentration of the Fc fragment blocking  (was about 100ug/ml the first time) and also to try an Fab fragment as a secondary, rather than the whole IgG we currently use.  Does anybody have any other suggestions as to what we might try to get on top of this problem?  I have only ever immunostained mammalian cells and tissues before, so this is my first time with bacteria.  The dyes I'm using (Cy3 and DAPI) photobleach very easily for some reason with these bugs compared with your average mammalian cells? 

  

Thanks for any suggestions, 

  

Jason 

  

Jason Palmer 
Histology Laboratory Coordinator 
O'Brien Institute 
42 Fitzroy St, Fitzroy Victoria 3065 
Australia 
tel +61 3 9288 4045 
fax +61 3 9416 0926 
email: jpalmer <@t> obi.edu.au