topic 6 Flashcards
what is SDS and what does it do?
SDS is a reagent used to denature proteins by the breaking of disulfide bonds and HB, while coating them in a uniformly negative charge
what compontes in SDS are used to break Disulfide bonds?
2-mercapetoethanol and DDT
what is an SDS page?
a method used to evaluate the purity of a protein. in this technique, the coated protines travel down the pos electrode, where HEAVY port are suspended and the top and the LIGHT prot can make their way down
test is based on MASS ONLY
what is the crude extract? how is it obtained
the cured extract contains all of the proteins found within a cell, but without the cell components. the cell is lysed via sonication to form a supernatant (solution with soluble proteins in it)
what is affinity chromatography
the tendency for proteins to be attracted to specific molecules which are attached to beads. a crude mixture will pass over the beads, and the proteins with a high affinity for the molecules will be attached and filtered from the other proteins. the protein of interest is then released using a concentrated solution of the attracting molecule
what is metal affinity chromatography?
the use of his-tags to bind to metal ions to purify a protein sample. his interacts with the metal ions, conserving the target protein in solution. the protein is then eluted suing a solution of imazidole or a low pH.
what is salting out?
the act of increasing the salinity of a protein-bearing solution to encourage the precipitation of protein. this is useful as each protein has a unique solubility factor while in an aqueous solution, so different levels of salt allow for the precipitation of different proteins.
what is size exclusion chromatography
the process of filtering proteins based n their size through the use a gel filtrate containing porous beads. small proteins are able to enter the beads and are forced to take a long windy path. larger beads are unable to enter the beads, so they pass through a quicker and more direct route. the large proteins are emerge first.
how is the purity of a sample tested using and SDS page?
a pure sample will exhibit one uniform, clean line at one specific wight, indicating the presence on one protein. there my by two proteins in the final product, but this will result in 2 distinct bands. As the purity of the protein solution increases, the quality of the SDS page increases too (less band and easier to read).
