Lecture 6: Analyzing Membranes Flashcards
1
Q
Fluorescence recovery after photobleaching (FRAP)
A
Technique to study movement of membrane components (proteins or lipids)
1
Q
Fluorescence recovery after photobleaching (FRAP) Steps:
A
- Label membrane component with a flourescent dye (eg: flourescent anitobody that recognizes a particular protein)
- Photobleach (remove fluorescence from a portion of the cell)
- Monitor reappearance of flourescence in the previous bleached portion. Rate of recovery of fluorescence is a measure of the rate of diffusion of the fluorescently labeled protein
2
Q
Examining protein size or expression levels with gel electrophoresis steps (4):
A
- Lyse the cells and collect the plasma membrane through mechanical disruption, freezing/thawing or hypotonic solution. Pellet1= membrane and membrane protein Supernatant1= aqueous buffer
- Isolate the peripheral membrane proteins using high salt. ions from the salt will compete with the charged amino acid of the peripheral memebrane proteins to disrtupt the noncovalent interactions with the membrane. Pellet 2= transmembrane protein. Supernatant2= peripheral membrane
- Isolate the transmembrane proteins with strong detergent (amphipathic). This makes the transmemnrane proteins soluble in aqueous solution. Supernatant3=Transmembrane proteins Pellet3=GPI
- Isolate GPI-anchored proteins by treatment with phosphatidylinositol-specific phospholipase. Pellet4= insoluble materials. Supernatant4= soluble materials
3
Q
Electrophoresis
A
Seperation of charged molecules by migration through an electric field
4
Q
Before we analyze the protein’s migration through an acrylamide gel, we need to —- the proteins by—–
A
-denature
- adding SDS which gives the proteins a uniform negative charge (disrupts protein folding)
5
Q
How does SDS work?
A
- SDS coat protein in a negative charge and unfold/break any noncovalent bonds holding it together through repulsion between bound SDS molecules
