Isotype controls

Some laboratories are still using isotype controls. It is not that these are worthless in all situations but you might consider skipping them.
Isotype controls are never the same reagents as the specific antibody (Ab). The chemistry of the reagent, the concentration, the purity, the efficiency of labeling, the solubility of the protein and the conjugate make using an isotype control not always valid. Especially when the preparations are not from the same company the differences between the preparations will be bigger. For instance, there are several forms of fluoresceine, antibodies are 'all' different in their constant regions which make the efficiency to bind Fc receptors different and preparations with a lot of free fluorochrome will lead to a higher background than purified preparations.

In our opinion there are two better controls:

	The more expensive one: add 10 x molar excess of the target protein to the specific Ab (regular concentration) before you add this to your sample. The signal in this sample will be due to binding of the fluorochrome to the cell or binding of the Ab to Fc receptors. This incubation should be negative.
	Instead of the isotype control; Compare your samples with the unstimulated version of the same sample (but with transport inhibitor). All positive events are due to the stimulation. This incubation does not have to be 100% negative!
	You also might consider a preincubation with the unlabeled from of the antibody to whether conjugation of the antibody is of influence on the aspecific binding of the antibody. This incubation should also be negative.

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Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005