Biacore sample preparation
The user should bring the Biacore sensor chip suitable for the planned work. A ligand (a compound that would be immobilized covalently to a chip surface through its NH2- groups must be dissolved in a buffer which does not contain primary amino groups (e.g ammonium ions, Tris, glycine, ethanolamine, triethylamine, glutathione etc). The desired final concentration of immobilized material should be not less than 10 micrograms/ml. In cases where the common Biacore buffer is not compatible, the user should choose a buffer suitable for his system. Buffers must be fresh and passed through 0.2micron filter.
ITC sample preparation
It is critical to optimize the starting concentration of the protein in the cell according to the expected dissociation constant. Furthermore, the titrant's concentration should be approximately 10 times higher than the protein concentration in the cell. Lastly, the cell requires at least 0.5ml of protein solution and the syringe about 80μl.
The experimental setup is quite simple: a syringe containing a "ligand" (either a small molecule ligand or an interacting protein) is titrated into a cell containing the protein solution. Critically, the buffers of both solutions must be identical. This can be achieved by dialysing both substances against the same buffer.
DSC sample preparation
The concentration of the protein should be 0.5-1mg/ml in 1ml. Criticaly, the buffers in the reference and sample cells must be identical. This can be achieved by disalysis of the sample against the buffer in the reference cell.
MST sample preparation
Protein (a compound that would be labeled) should be in a buffer that does not contain primary amines (e.g., ammonium ions, Tris, glycine, ethanolamine, triethylamine, glutathione) or imidazole. All of these substances will significantly reduce protein labeling. Also, His Tag can be labelled. Concentration of protein for labeling should be not less than 5 µM.
Unlabelled compound should be in the range of 10-20 of the expected dissociation constant.