Sathi, You could use an biotinylated anti-FITC antibody, followed by SA-PE. You will be able to get a good boost with that (upto 1 log in intensity). Bio-anti-FITC is available with most commercial vendors (DAKO, caltag, pharmingen I assume). However, I am working with a signal amplification technology which could be easily applied onto your system. Here is a reference to the abstract. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_u ids=11438455&dopt=Abstract) About using a second FITC-labeled antibody, it may be OK to use it only if it is used as a positive marker. (i.e. you will be looking at double stained cells at the end of the experiment). This again depends on - how much peptide-FITC is bound to the cells? is it as bright as the second FITC-labeled antibody or not? Does it resolve as two different peaks/populations based on relative intensities etc., good luck, Ragoo *********************************************************** Arumugham Raghunathan, Ph.D Group Leader, Flow Cytometry Group Cellular Analysis Section Molecular Staging Inc., 300 George Street, Suite 701 New Haven, CT 06510 Tel# 203-772-5074 Fax# 203-776-5278 http://www.molecularstaging.com ************************************************************* -----Original Message----- From: Sathiyaseelan [mailto:t.sathi@VERIZON.NET] Sent: Friday, May 03, 2002 12:34 AM To: cyto-inbox Subject: Anti FITC antibody Hi I have a FITC-conjugated peptide that binds to the surface of a cell line and gives only a moderate intensity of FL1 signal (in 2nd decade) when analyzed by flow cytometry. Unfortunately, I do not have a biotin-labelled peptide in order to use a PE-strepavidin second reagent to amplify the signal. Could any one tell me if there is a possibility to use an anti-FITC antibody conjugated to PE as a second reagent to amplify the fluorescent signal. How much signal amplification I could possibly achieve using this PE conjugated-anti-FITC antibody method and can I use another FITC-labeled antibody to label another surface antigen simultaneously? Does anyone know a source of this antibody? I would appreciate all useful info and help on this. Thanks SATHI
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