Protein Analysis sample

Sample Preparation for submission

Biacore sample preparation

A customer has to 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 contained primary amino groups (Tris, glycine etc). The desirable final concentration of immobilized material should be not less then 10 mg/ml. In the case the common Bia buffer is not compatible, a customer has to choose a running buffer suitable for his system.

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 needs to be reasonably soluble, since an approximately 10 times higher concentration than for the protein in the cell is required. 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 highly temperature controlled cell containing the protein solution. Critically, the buffers of both solutions need to be identical, which can be achieved by dialysis of both substances against the same buffer. When the two proteins or the protein and the ligand interact, heat is released or absorbed. When the protein in the cell becomes saturated with added ligand or protein, the heat signal will become smaller and smaller until only the background heat of dilution is observed. This raw ITC data can be fitted and ΔH and ΔS can be extracted.

DSC sample preparation

Critically, the buffers in reference and sample cells need to be identical, which can be achieved by dialysis of sample against the buffer in 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. Concentration of protein for labeling should be not less than 5 µM.

Unlabelled compound should be in the range of ± factor 10-20 of the expected dissociation constant.