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Fab fragments are a good idea, but getting them can be very time-consuming and expensive.  Some companies offer this service, but the quality is highly variable - naming no names, we've had some total failures to produce labelled Fab that still binds.  In our own laboratory, producing Fab from IgG varies enormously; MHC class II works well, CD74 seems to be impossible.  You need to make a number of test runs with variable conditions, so shedloads of IgG may be required.

If you have to use a 514nm excitation, can I put in a word for Oregon Green 514 (no commercial interest).  This can label whole antibody up to ~25probes per IgG with no self-quenching, and on the samples we made using MHC antibodies there was no aggregation and the binding was only slightly compromised.

Ian

----------------------------Dr. I.E.G.Morrison       [log in to unmask]
                            Dept.Biological Sciences, University of Essex
                            Wivenhoe Park, Colchester, Essex CO4 3SQ
----------------------------Tel: 01206-872246           Fax: 01206-872592--------------------------