Analyzing Membranes

Question 1

What are the two ways to study membrane protiens

Answer 1

Frap (fluorescence recovery after photo bleaching)
Gel electrophoresis

Question 2

What is FRAP

Answer 2

A method used to study membrane dynamics in vivo (living) while the proteins are still in the cell

Question 3

What is gel electrophoresis

What needs to happen

Answer 3

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

Question 4

What are the dynamics of membrane protiens (many ways they can move)

Answer 4

1. Random diffusion (can move freely)
2. Immobilized (no movement)
3. Motor protiens have a certain movement
4. Can be restricted by other integral membrane protiens
5. Can be restricted by membrane skeleton protiens
6. Can be restrained by extracellular materials

Question 5

What happens in step one of FRAP

Answer 5

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.

Question 6

What happens in step 2 of FRAP

Answer 6

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

Question 7

What happens in step 3 of FRAP

Answer 7

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

Question 8

If in Frap we see less fluorescence recovery what does this mean

Answer 8

The protein is least able to move

Question 9

What is the first step to separating protiens to analuze them through electrophoresis

Answer 9

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)

Question 10

Is step 1 of separating membrane protien, where are the membrane and its accosiated membrane protiens

Answer 10

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

Question 11

What is step 2 to separating protiens to analuze them through electrophoresis

Answer 11

Isolate the PERIPHERAL membrane protiens by centrifuging pellet 1 in HIGH SALT concentration

This makes pellet 2 and supernatent 2

Question 12

In step 2 to separating protiens to analuze them through electrophoresis why do we use high salt concentration

Answer 12

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

Question 13

In step 2 to separating protiens to analuze them through electrophoresis what is in pellet 2 and in supernatent two

Answer 13

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

Question 14

What is step 3 to separating protiens to analuze them through electrophoresis

Answer 14

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

Question 15

In step 3 of separating protiens to analuze them through electrophoresis what does the non ionic detergent do?

Answer 15

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

Question 16

In step 3 to separating protiens to analuze them through electrophoresis what is in pellet 3 and supernatent 3

Answer 16

The now soluble transmembrane protiens are in supernatent 3

GPI anchored lipid proteins (extracellular side of membrane) are in pellet 3

Question 17

Where are gpi achored protiens usually found and what are they linked to

Answer 17

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

Question 18

What is step 4 to separating protiens to analuze them through electrophoresis

Answer 18

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

Question 19

In step 4 of separating protiens to analuze them through electrophoresis what is in supernatent 4

Answer 19

The gpi anchored porotiens

Question 20

When doing the whole isolating embrace protiens process why do we do it in the specific order

Answer 20

This releases the weakly associated protiens first

Question 21

What is electrophoresis

Answer 21

Departing charged molecules through their migration in an electric field

Question 22

What is polyacrylamide gel electrophoresis (PAGE)

Answer 22

The protiens migrate through a gel matrix made of cross linked acrylamide polymers

Question 23

What do we need to do before analyzing the proteins migration through PAGE

Answer 23

First we need to denature the protiens and apply a negative charge

Question 24

How are protiens denatured in order to be analyzed by PAGE based only on size

Answer 24

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

Question 25

After protiens have been denatured and negatively charged, what are the next steps to the SDS PAGE

Answer 25

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

Question 26

In SDS PAGE how do we visualize the protiens in the gel and estimate their sizes

Answer 26

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

Question 27

What can SDS PAGE tell us

Answer 27

Size of protiens (larger, higher)

Protien concentration/abundance (bigger band, more abundant)