Question

From the following list of laboratory materials, choose which three reagents would have been required to prepare the [Human sex hormone binding globulin] hSHBG sample for the separation step of isoelectric focusing with a commercial 1PG strip (pH 4-7).

Give reasons for your selection.

Laboratory materials:

ethanol, urea, SDS, reducing agent (e.g. dithiothreitol), sodium chloride, hydrochloric acid, a non-ionic detergent (e.g. Triton X-100, CHAPS), dimethyl sulfoxide, agarose

Answer 1

Isoelectric focusing is a technique which allows the separation of proteins on the basis of their isoelectric points (the pH at which a protein has no net charge). The sample preparation for isoelectric focusing requires these three reagents :

Urea: Urea is added while sample preparation because it acts as a solubilizing agent and prevents the aggregation of protein while the procedure is ongoing. It also denatures the protein by disturbing the hydrogen bonding present in the protein and had maintained its globular 3-dimensional structure. If the protein gets aggregated, its mobility in the procedure would be decreased and hence will affect its separation.

Reducing agent (e.g. dithiothreitol): This is included to disrupt the disulfide linkage present in the protein. This reagent actually a reducing agent will reduce the disulphides(-S-S-) to thiol groups (-SH) again preventing the protein to attain its secondary structure.

Non-ionic detergent (e.g. Triton X-100, CHAPS): Non-ionic detergents are used to aid in protein solubilization and its denaturation into straight chains. The detergent for this technique has to be non-ionic because the actual charge on the protein has to be preserved as this only is the basis of protein separation in this process.

Ionic detergent like SDS cannot be used because it results in negative charge on the protein and actual charge on the protein is lost.