Module 3

Question 1

7 Steps to Isolating a Protein

Answer 1

1) Develop a good assay
2) Select a protein source
3) Break open cells
4) Solubilize protein
5) Stabilize protein
6) Fractionate
7) Determine purity

Question 2

Step 1: Protein Assay

Answer 2

A method of detecting for the presence of a specific protein, it must be unique to the protein of interest so other proteins are not mistaken

Question 3

Step 2/3: Protein Source & Extraction

Answer 3

1) Protein should be easily obtained
2) Present in large amounts
3) Low in proteins that have similar characteristics
4) Low in proteases that may destroy the protein of interest
5) Bio-membranes are broken down so protein can be extracted

Question 4

Step 4: Protein Solubilization

Answer 4

1) The protein of interest must be solubilized for further testing.
2) Cytosolic and secreted proteins are expected to be soluble
3) Transmembrane and membrane-associated proteins are not. This is because membrane proteins are often amphipathic, meaning the proteins will be difficult to isolate.
4) Protein solubility is affected by pH, salt concentration, and detergents which help stabilize molecular interactions

Question 5

Step 5: Protein Stabilization - What are the important parameters?

Answer 5

You should try to maintain the non-covalent interactions that are stabilizing the folded conformation.

1) Temperature
2) Protease Inhibitors
3) Ligands
4) Salts
5) Metal Ions
6) Concentration
7) pH

Question 6

Step 6: Fractionation - How do proteins differ from one another?

Answer 6

1) The process of separating proteins into different groups or fractions
2) Multiple fractionation techniques will be required
3) Proteins vary in: size, polarity, charge, solubility, shape

Question 7

Explain Differential Centrifugation

Answer 7

1) 1000 CF: nuclei, chloroplast
2) 10 000 CF: mitochondria
3) 100 000: ER, Golgi, lysosomes, peroxisomes
4) Remaining: cytosolic proteins

Question 8

Ion Exchange Chromatography

Answer 8

1) Beads are charged, either positive or negative
2) Respective charged proteins will either be attracted to the beads or fall quickly
3) Elute the protein of interest using a salt solution or warm wash

Question 9

Gil Filtration Chromatography

Answer 9

1) Beads have very small holes or cavities in them
2) Small proteins are trapped in the cavities and large proteins flow straight past
3) Remove the beads and wash them or spin the beads slowly to dislodge the proteins

Question 10

Affinity Chromatography

Answer 10

1) Beads are covalently attached to an antibody
2) Separates proteins based upon specificity of binding to another molecule
3) Protein of interest will stay on the column due to non-covalent interactions with antibody
4) Remove by changing pH, raising temperature, increasing salt concentration
5) EX: The Ras protein will bind to GTP in this type of fractionation

Question 11

SDS-PAGE Electrophoresis

Answer 11

1) Intentionally denaturing the protein (eliminates shape)
2) Prepare the sample by using sodium dodecylsulphate (SDS) to denature proteins and coat them
3) All proteins will have a negative charge, eliminating effects of shape and charge
4) Proteins are loaded onto a polyacrylamide gel
5) Electrical current causes all negatively charged protein to move towards the positive end of the polyacrylamide gel
6) Based on molecular weight, large proteins move slowly will small proteins move quickly
7) Also, use a Western Blot to transfer proteins to a membrane with specific antibodies to identify the protein of interest

Question 12

Step 7: Purification

Answer 12

Specific activity is total enzyme (or protein) activity divided by the total amount of protein in solution. By the end, there’s a decrease in volume, total proteins, and activity but an increase in specific activity of the protein of interest

Question 13

Light Microscopy

Answer 13

Uses visible light, conventional and fluorescent

Question 14

Electron Microscopy

Answer 14

Uses beams of electrons, transmission and scanning, less than 200nm in size

Question 15

Resolution (D)

Answer 15

1) The minimum distance between two objects that can be distinguished from one another
2) Smaller value of D means the resolution is better and you can see more details
3) Calculated using wavelength of the source of illumination (lambda) and the numerical aperture (NA)
4) Smaller wavelength and larger NA will improve resolution, as D gets smaller

Question 16

Brightfield Microscopy

Answer 16

1) Samples can be live or fixed, stained or unstained
2) Single cells, tissues

Question 17

Nomarski Microscopy vs Phase Contrast Microscopy

Answer 17

1) Phase Contrast favours clear visualization of internal cellular structures
2) Nomarski produces clearer, sharper images of edges and surfaces of cellular structures
3) Both reply upon enhancing the inherent difference in density of different regions of the specimen
4) Both can visualize live specimens

Question 18

Immunofluorescence Microscopy

Answer 18

1) Molecules of interest are stained directly with fluorescent dyes or tagged with fluorescent antibodies
2) Antibody recognizes molecule or antigen of interest, then, a secondary antibody attaches, then a fluorophore, which is excited by UV light
3) DAPI is a blue stain used for DNA

Question 19

Green Fluorescent Protein (GFP)

Answer 19

1) A naturally-derived fluorescent protein that was originally discovered in jelly fish
2) Create a gene fusion between the gene coding for the protein of interest and the gene coding for GFP, allowing the gene to express the fluorescent fusion protein
3) GFP can be used for live cells

Question 20

Confocal Scanning Microscopy

Answer 20

1) Obtains high-resolution images from fluorescently labeled samples
2) Uses optical sections, exciting only the fluorophores in a thin section of the sample with a specialized laser
3) EX: In BPAE cells, the nucleus is stained blue with DAPI, action is tagged in green, and the Golgi apparatus is tagged in red

Question 21

Deconvolution Microscopy

Answer 21

a) Uses traditional fluorescence microscopy to produce a clear image
b) Computer algorithms subtract the fluorescence out of focus above and below the focal plane of the image

Question 22

Transmission Electron Microscopy (TEM)

Answer 22

1) Increased magnification of images by improving resolution
2) Limit of resolution of 0.1nm
3) The beam is directed through a thinly sliced specimen to form an image

Question 23

Scanning Electron Microscopy (SEM)

Answer 23

1) The beam is focused over the surface of the specimen that has been coated with a thin layer of metal
2) Produced an image of the 3D surface morphology

Question 24

What are some of the characteristics and functions of bio-membranes?

Answer 24

1) Selectively permeable due to transport channels
2) Hold proteins that can mediate cell-to-cell interactions, like signalling
3) Flexible and dynamic, can change shape without breaking

Question 25

Cytosolic Face vs Exoplasmic Face vs Lumenal Face

Answer 25

Cytosolic Face: faces the cytosol or inside of the cell Exoplasmic Face: faces outside of the cell Lumenal Face: faces the interior of the organelle

Question 26

Biomembrane Bilayer Structure

Answer 26

1) TEM, stained with osmium tetroxide, reveals the membrane bilayer 2) Basic structural unit of biomembranes is the phospholipid, an amphipathic molecule with a hydrophilic (polar) head and hydrophobic tail 3) In smaller concentrations, phospholipids associate into a micelle, a sealed ball but at higher concentrations, they spontaneously assemble into the bilayer 4) Composed of two hydrophobic fatty acids (hydrophobic) linked to glycerol with a phosphate group (hydrophilic)

Question 27

Types of Membrane-associated Proteins

Answer 27

1) Integral Membrane Proteins: span across the phospholipid bilayer 2) Lipid-Anchored Proteins: anchored in one leaflet 3) Peripheral Proteins: attached to the polar surface of the membrane or indirectly via other proteins

Question 28

T or F: Biomembranes can fuse with one another, can be deformed without tearing, and can change in shape to accompany any cell movements, like when a cell is undergoing cell division. Proteins and phospholipids can move laterally through the membrane

Answer 28

True

Question 29

Fluid Mosaic Model

Answer 29

1) We refer to the biomembrane as a Fluid Mosaic Model 2) Fluid because the membrane components move laterally or sideways throughout the membrane 3) Mosaic because it's made of many different kind of macromolecules

Question 30

FRAP

Answer 30

1) Fluorescence Recovery After Photobleaching 2) Fluorescent molecules attached to proteins of interest are bleached and saturated, no longer being able to fluoresce 3) A small patch of fluorophores are bleached 4) Depending on fluidity of the membrane, fluorescence in the bleached area can be recovered due to other non-bleached fluorophores moving into the bleached area 5) Demonstrates that membranes and macromolecules within them are indeed fluid

Question 31

T or F: In a FRAP experiment to calculate fluidity, there was initially 3000 units of fluorescence. However, after bleaching, this dropped to 1000 units, which gradually grew to reach a maximum of 2000 units. This showed that 50% of membrane receptor proteins are mobile in the membrane and the other 50% are immobile, since 50% of original fluorescence was recovered.

Answer 31

True

Question 32

What factors can influence the fluidity of the biomembrane?

Answer 32

1) LESS FLUID: Saturated fatty acid tails are straight with no double carbon bonds 2) MORE FLUID: Unsaturated fatty acids are bent with double carbon bonds 3) LESS FLUID: Longer fatty acids can pack together more tightly (18 carbons per chain) 4) MORE FLUID: Shorter fatty acids don't pack tightly (16) 5) More cholesterol decreases fluidity, less increases fluidity, it helps prevent extremes and regulates fluidity

Question 33

Is there movement between leaflets of the biomembrane?

Answer 33

1) It is difficult to move proteins from one leaflet to another because the polar head group of each phospholipid would need to pass through the hydrophobic core 2) It is possible through flippases

Question 34

Single-Pass Transmembrane Protein, Example?

Answer 34

1) A type of integral membrane protein 2) Has a single hydrophobic alpha helix that spans the phospholipid bilayer 3) EX: Glycophorin A is found on human red blood cells, is a homodimer, with each part of the dimer being a single pass transmembrane protein

Question 35

Multi-Pass Transmembrane Protein, Example?

Answer 35

1) They pass through membranes many different times 2) EX: Bacteriorhodopsin is a typical seven transmembrane spanning protein, with seven alpha-helices that interact with one another to form a transmembrane domain

Question 36

Beta-Barrels

Answer 36

1) Exterior is hydrophobic so it can interact with the hydrophobic membrane 2) Interior is hydrophilic core 3) EX: porins found in bacteria cells, chloroplasts, and mitochondria

Question 37

Channels

Answer 37

1) Collection of alpha-helices 2) EX: aquaporin that creates a hydrophilic channel for water movement

Question 38

Lipid-Anchored Proteins

Answer 38

1) Associated with one leaflet of the membrane by covalently attached lipid modifications 2) Modified by acylation (attaches lipid anchor to N-terminus) or prenylation (attaches lipid anchor to C-terminus) 3) Some have a GPI anchor