Product Number CM7xxxx
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CM71005: SepSphere™ Small Molecule Amine Immobilization Kit via Glyoxyl
How do I incubate the immobilized small molecule agarose beads with cell lysates?
This kit is for preparing small molecules immobilized onto agarose beads. After the washing step in B11, the resin immobilized with the small molecule is ready for usage. Just add the cell lysates directly to the column with the bottom plugged if you are using all the resin in a column format or take some resin out and add to the cell lysates in a centrifuge tube/spin column. The amount of resin you will use depends on the binding capacity of your small molecule and the amount of protein you would like to capture. Nutate the column at RT for a few hours or overnight. The time for incubation depends on how strong and fast your small molecule can bind to the protein. After incubation, you will wash with water or PBS similar to step B11.
How do I dissociate the binding between my small molecule and the protein?
When you are trying to dissociate your small molecule and protein, you can change the pH (low or high), use high salt (ionic strength), or use detergents or chaotropic agents that can denature one or both of the molecules. For Western blotting or LC-MS, your protein doesn’t need to be active (can be denatured). Unfortunately, which buffer to use will depend entirely on the small molecule and protein interaction. You can figure it out by trying a few conditions. You may be able to find some of these conditions in the literature. I would suggest trying an acidic buffer first, such as 0.1M sodium citrate (pH 3.3) or 0.1M glycine buffer. These are commonly used for disrupting protein A and antibody interaction. For LC-MS, try to avoid using any detergent as this will suppress the signal.
How can I make bead-only samples for control?
If you want a simple control, you can use any of the non-modified agarose beads. If you want to use an exact control, you may need to use another kit to prepare a control with a non-binding small molecule amine.