Tetramer Protocol: Tetramerization

General guidelines on tetramerization

The following information is a generalization of how much labeled streptavidin to add to the biotinylated monomers at teh last stage of the tetramer preparation.  The actual amount of streptavidin to be added depends both on the molecular weight of the monomer as well as the percent biotinylation.  In the Altman Lab, this amount will be calculated for you in the "JDA Tetramer stocks" database and given as a total volume to be added to the monomer solution.  As such, it is necessary to divide this amount by ten to arrive at the amount to be added per time interval.

The procedure of adding the streptavidin over 10 time intervals is designed to maximize tetramer formation.  At early additions, the biotinylated MHC will be in excess and will saturate all of the avidin.  At the conclusion of the streptavidin addition, there may be an excess of avidin, however, the assumption is that most of the MHC will have gone into tetramers.  If the streptavidin was added at one time and was in excess, the resulting solution would be more likely to exist as a mixture of monomers/dimers/trimers.

This approach is summarized in the figure below.

Comments on stretpavidin addition protocol

Theory

Our standard storage conditions for biotinylated monomer stocks are 100 µl at 2 mg/ml, for a total of 200 µg of MHC protein. For the purposes of calculation of how much streptavidin to add, we usually assume 100% biotinylation. Even if we are wrong, the worst that can happen is that you'll waste a bit of streptavidin; it probably won't add much to the background.

We exclusively use streptavidin-PE and streptavidin-APC from Molecular Probes. These are sold at a concentration of 1 mg/ml, which refers to the total weight of the conjugate. We assume a 1:1 ratio of streptavidin to phycobiliprotein, and we have calculated the concentration of biotin binding sites in these stocks. This is an estimate, but the addition protocol is devised to provide good results even if our estimates are off. In practice, we find that this procedure is very robust.

Tetramerization Protocol

The addition of streptavidin to the biotinylated monomers is performed at room temperature, with the samples kept in the dark between additions (usually in a drawer). The protocol will probably also work if the samples are kept on ice. The key is to add the streptavidin in small subaliquots, and to mix thoroughly (but gently) after each addition. The streptavidin volumes listed below are appropriate for 200 µg aliquots of biotinylated MHC.