Question

A new graduate student is performing a His-tag protein purification. They create a lysis buffer, wash buffer and elution buffer, with respective increasing concentrations of imidazole. You accidently use the elution buffer to lyse your cells, but decide to perform the His-purification anyways, and run your finished sample on an SDS-PAGE gel stained with instablue dye. What do you expect the end result to be and why (2)?

Answer 1

The end result will be seeing lots of non specific protein on SDS -PAGE , a quality near to crude lysate. His tag proteins are purified using Ni-Nta columns, wherein the Imidazole competes with target protein to bind with the Nickel in the column. Elution buffers have a high concentration of imidazole therefore if its used in thge begning itself that is instead of lysis buffer, most of the proteins along with thhe target protein will wash out in the sample during elution. Also the protein yield will be lesser because lysis buffers typically use lysozymes and other agents to promote release of protein into the crude lysate, and elution buffer will not have these agents So whatever protein that manages to get released will mostly elute out unbound to the column.