The easiest way to go is using FITC-anti-IgG. This works very well, usually gives some amplification of signal, and there are plenty of commercially available anti-Ig's. However, dependent upon the cells you are using or the experiment, there are several important considerations. 1) If you are looking at cells that are or contain B lymphocytes, you need to make sure that your sandwich antibody is not recognizing the surface Ig on the B cells. So if you are looking at mouse spleen cells and you use a anti-rat IgG-FITC, then you have to make sure that it is specific for rat Ig and not mouse. This is difficult to obtain, unless you use a monoclonal mouse anti-rat. 2) Be very careful using sandwich antibodies when you are doing multicolor flow cytometry. Often the other antibody you are using may be of the same strain as the first and you will get cross reactions. Blocking with unlabeled antibodies may only partially alleviate those cross reactions. If the above issues are difficult to solve in your particular experimental situation, then you can label the antibody yourself, but again there are issues to be aware of: 1) You need to determine whether a stabilizing protein has been added to the antibody you bought. If so, it will interfere with labeling and you cannot do the conjugation, unless the antibody is repurified. 2) Labeling small amounts of antibody can be done, but you need to use microdialysis chambers (Pierce sells these) in order to perform the dialysis steps, or you will loose too much protein. Also, it is more critical to have the antibody at a higher concentrations, i.e.. 1 mg/ml, than to have a lot of protein. Hope this helps. Kevin L. Holmes, Ph.D. Head, Flow cytometry Unit Office of the Scientific Director Bldg 7, Room 01 NIAID, NIH Email: kholmes@atlas.niaid.nih.gov ---------- From: Dave Novo[SMTP:novod@muss.cis.mcmaster.ca] Sent: Thursday, March 27, 1997 12:06AM To: cyto-inbox Subject: conjugation vs. sandwich Hello everyone, I have to buy an antibody for which it seems that there is no company selling one pre-conjugated to a dye. I was wondering what people thought of the relative merits of conjugating FITC to the antibody myself vs. buying a generic anti IgG-FITC premade conjugate and using a sandwich type technique. What are the pro's and cons of the various ideas? Thanks in advance, Dave *********************************************************************** **** Deep thought of the day: If the local 7-11 is open 24 hours a day - why are there locks on the doors? *********************************************************************** ****
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