Lecture 7 - Purification, Detection, and Characterization of Proteins I Flashcards
Provide a summary of rate-zonal centrifugation.
For this technique, you layer a sample on top of a density gradient before exposing the mixture to a centrifugal force. The particles will settle according to mass. You can then collect fractions from your mixture to isolate different components of your original sample based on their mass.
Provide a summary of differential centrifugation.
For this technique, you pour a sample into a tube and centrifuge it. Heavy particles will settle in the bottom of the tube as pellets. You can decant the supernatant (liquid) out to isolate the pellets. You can also recentrifuged the supernatant at a higher speed to make pellets of heavier components of the sample.
Provide a summary of SDS-PAGE.
SDS-polyacrylamide gel electrophoresis separates proteins based on their mass. You subject proteins to an electric field to separate them by their ability to migrate through the gel towards one of the poles. To do this, you boil proteins in the presence of SDS (sodium dodecyl sulfate) to mess up their hydrophobic interactions and denature them before placing them in a polyacrylamide gel, a polymer that’s been cross-linked so that there are little channels. When the electric field is applied, all proteins possess the same electromotor force, but the larger proteins movement is impeded by their size. As such, small proteins migrate far and large proteins migrate short distance.
Provide a summary of immunoblotting.
Immunoblotting combines SDS-PAGE with a subsequent step that’s based on an antibody that recognizes your protein. You put your SDS-PAGE gel and put it on a solid-state matrix (nitrocellulose or nylon) to preserve the relative positions of the proteins. You mix the matrix with your antibody and wash away any excess. You then mix with a second antibody that gives off light and recognizes the first antibody. You then wash the membrane and perform a reaction in order to see exactly where the proteins are.
Which type of blot analyzes RNA?
Northern
Which type of blot analyzes DNA?
Southern
Which type of blot analyzes proteins?
Western
Provide a summary of 2D gel electrophoresis.
2D gel electrophoresis separates proteins based on weight and charge. First, you run a sample of proteins through a pH gradient (isoelectronic focusing) established in a gel-like substance using ampholytes with an applied electric field. The proteins migrate until they reach their isoelectronic point. You then apply a field in the other axis to separate the proteins by size (like in normal SDS-PAGE).
Provide a summary of gel filtration or size exclusion chromatography.
Size exclusion chromatography separates proteins by size by running them through a chromatography column containing an array made up of agarose beads or polydex stands. Small proteins tend to get caught up in the column (they go in all the cracks and take the long way) while large proteins flow through without being impeded. If you collect separate eluates into fractions, the first fractions will contain the large proteins while the last fractions will contain the small proteins.
Provide a summary of ion exchange chromatography.
Ion exchange chromatography is performed similarly to size exclusion chromatography, except the beads are given positively or negatively charged groups. Proteins with a like charge to the beads will elute first while proteins with an opposite charge will elute last (once a salt is added to outcompete the proteins’ attraction to the beads).
Provide a summary of affinity chromatography.
Affinity chromatography utilizes an antibody bound to the beads to separate out specific proteins. Proteins that do not correspond to the antibody will elute first while proteins that do correspond will elute last (after introducing a low pH buffer to remove them from their antibodies).
