I've done quite a lot of fluorochrome labeling, and all three of the approaches you described will help. The first two are obviously the ones that apply to the antibody you presently have. The scale down process is possible, but efficiency tends to be low, especially if you don't have enough to work with a few different conditions. Concentration is probably the best way to go; especially with a starting solution as low as you described, denaturation really shouldn't be a problem, and you'll have no problem getting it concentrated to 2mg/ml in a spin column. The one thing that is best avoided is trying to conjugate at the wrong concentration; with the conditions you describe, there would be a 20X increase in the molar ratios of chrome/antibody, and the likelihood that you'll end up with a badly denatured antibody is high. Even a 2-3X change in molar ratios can be detrimental with some antibodies, depending on the number and location of conjugation sites. Howard >Dear Scientists, > >I was wondering if someone could give me direction on fluorochrome labeling >of a Mab. The labeling kit I would like to use requires 2mg/ml starting Ab >concentration while the Mab I need to use is only available in a 100ug/ml >concentration. Would it be wiser to 1)try to concentrate the MAb through >say a spin column, 2)scale-down the labeling kit to fit the Ab concentration >(quite a scale-down), or 3)try the experiment with a polyclonal Ab available >in the proper concentration then spend a lot of time testing >cross-reactivity? The MAb I need to use is E. coli O157:H7, perhaps someone >knows of a supplier who produces greater concentrations. I am relatively >new to the field of Ab labeling so any feedback will be greatly appreciated. >Thank you for your time. > >Sincerely, >Amanda Votaw >Research Scientist >University of Wyoming -- Howard T. Petrie, Ph.D. Head, Laboratory of Developmental Immunology Memorial Sloan-Kettering Cancer Center Box 341, 1275 York Avenue New York, NY 10021 phone (212)639-2149 fax (212)794-4019
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:22 EST