Analyzing Membranes Flashcards
What are the two ways to study membrane protiens
Frap (fluorescence recovery after photo bleaching)
Gel electrophoresis
What is FRAP
A method used to study membrane dynamics in vivo (living) while the proteins are still in the cell
What is gel electrophoresis
What needs to happen
Looking at the protiens size or expression in vitro (glass)
This means the protiens are examined outside the cell and needs to be isolated from the membrane before we can study it
What are the dynamics of membrane protiens (many ways they can move)
- Random diffusion (can move freely)
- Immobilized (no movement)
- Motor protiens have a certain movement
- Can be restricted by other integral membrane protiens
- Can be restricted by membrane skeleton protiens
- Can be restrained by extracellular materials
What happens in step one of FRAP
To study the movement of the protiens in the cell
We first label the protein we’re interested in with a fluorescent antibody that binds to that specific protein.
What happens in step 2 of FRAP
After the protein’s fluoresced, we photobleach one part of the cell that had the fluorescent protiens. Bleached around a 1micrometer diameter hole
The photo bleaching removes the fluorescence
What happens in step 3 of FRAP
Need to examine the reappearance of fluorescence in the portion of the cell you bleached
The rate of recovery of the fluorescence tells you the rate of diffusion of the labeled protien
If the protien is mobile, with time it will move around the cell to fill that hole and fluorescence will come back
If in Frap we see less fluorescence recovery what does this mean
The protein is least able to move
What is the first step to separating protiens to analuze them through electrophoresis
First lyse the cells and collect their plasma membrane through mechanical disruption, freezing/thawing or a hypotonic solution
Once cells are lysed you centrifuge them to form supranatent 1 (soluble) and pellet 1 (insoluble material)
Is step 1 of separating membrane protien, where are the membrane and its accosiated membrane protiens
In the pellet 1. This is because pellet one is insoluble and all of the membrane had lipids which are also nonpolar so insoluble
Because the membrane goes to pellet all of the proteins attached to it go there as well
What is step 2 to separating protiens to analuze them through electrophoresis
Isolate the PERIPHERAL membrane protiens by centrifuging pellet 1 in HIGH SALT concentration
This makes pellet 2 and supernatent 2
In step 2 to separating protiens to analuze them through electrophoresis why do we use high salt concentration
The ions of the salt compete with the charged (polar) amino acids of the peripheral membrane protiens (since they’re hydrophilic)
This disrupts the non covalent interactions of the protiens with the membrane and releases them from it
In step 2 to separating protiens to analuze them through electrophoresis what is in pellet 2 and in supernatent two
Since we used high salt to remove the soluble peripheral membrane protiens, these proteins are in supernatent 2 (soluble)
The transmembrane protiens are in pellet 2
What is step 3 to separating protiens to analuze them through electrophoresis
Isolate the transmembrane protiens from pellet 2 by centrifuging it with a strong detergent that is amphipathic
Has to have polar and non polar ends
This makes supernatent 3 and pellet 3
In step 3 of separating protiens to analuze them through electrophoresis what does the non ionic detergent do?
Since the detergent is amphipathic it acts like a phospholipid and attaches to the transmembrane protiens
This makes the protiens stable and soluble in an aqueous solution
In step 3 to separating protiens to analuze them through electrophoresis what is in pellet 3 and supernatent 3
The now soluble transmembrane protiens are in supernatent 3
GPI anchored lipid proteins (extracellular side of membrane) are in pellet 3
Where are gpi achored protiens usually found and what are they linked to
In the detergent resistant portions of the membrane that are rich in cholesterol and sphingolipids
Linked to phosphatidylinositol in the membrane by and oligosaccharide sugar
What is step 4 to separating protiens to analuze them through electrophoresis
Isolating the gpi anchored proteins by using PI-PLC
Forms supernatant 4 and pellet 4
That’s an enzyme that cleaves the bond of the sugar from the phosphatidylinositol in the membrane to release the protien
In step 4 of separating protiens to analuze them through electrophoresis what is in supernatent 4
The gpi anchored porotiens
When doing the whole isolating embrace protiens process why do we do it in the specific order
This releases the weakly associated protiens first
What is electrophoresis
Departing charged molecules through their migration in an electric field
What is polyacrylamide gel electrophoresis (PAGE)
The protiens migrate through a gel matrix made of cross linked acrylamide polymers
What do we need to do before analyzing the proteins migration through PAGE
First we need to denature the protiens and apply a negative charge
How are protiens denatured in order to be analyzed by PAGE based only on size
Add SDS (negatively charged amphipathic detergent)
This denatures the proteins from it’s 3d structure (the repulsion of bound SDS molecules breaks the protiens non convent bonds like hydrogen and ionic bonds
and applies a uniform negative charge
After protiens have been denatured and negatively charged, what are the next steps to the SDS PAGE
the protien is mixed with tracking dye which help in loading, the protiens are loaded on to the gel
Then the electric current is applied and protons migrate to postive end
That tracking dye migrates all to the very bottom of the gel
In SDS PAGE how do we visualize the protiens in the gel and estimate their sizes
To visualize the protiens we use a coomasie blue dye which helps us to see the bands on the gel
The find the size of the protiens, we load a molecular weight marker/ ladder into the first lane which measure the mass in kDa
What can SDS PAGE tell us
Size of protiens (larger, higher)
Protien concentration/abundance (bigger band, more abundant)
