Protein Extraction

Question 1

What are the forms of protein extraction?

Answer 1

Sonication, French press, osmotic shock, digestion, detergents, homogenization

Question 2

What is sonication?

Answer 2

metal probe vibrates at high frequencies which creates waves through the test tube; this breaks apart the cells; vibrations create heat which may denature enzymes; may also require single cells because vibrations not penetrate tissues

Question 3

What is french press?

Answer 3

pressure; a threaded piston spins and pushes down the liquid, creating pressure in the solution; the pressure will then be released very quickly, causing the cells to burst open; this is good for eukaryotic cells when you want to keep the nuclei and organelles in tact

Question 4

What is an anion exchange column?

Answer 4

Where the stationary phase is + and the target compounds are negative; they will interact more strongly with the stationary phase and elute in the later volumes

Question 5

What is a cation exchange column?

Answer 5

Where the stationary phase is -, and the target compounds are positive; they will interact more strongly with the stationary phase and elute in the later volumes

Question 6

What is osmotic shock?

Answer 6

Cells are introduced into a hypo-osmotic solution, which causes them to swell and burst open, releasing their contents; works best on cells without cell walls

Question 7

What is digestion

Answer 7

Enzymes digest the cells

Question 8

What are detergents?

Answer 8

Break down the cell membrane to release the cell’s contents

Question 9

What is homogenization?

Answer 9

Using force to break things apart, such as a blender; necessary if we have multicellular samples, such as tissues

Question 10

What is activity?

Answer 10

How many Units of enzyme per mL (concentration)

Question 11

What is specific activity?

Answer 11

The amount of Units of enzyme per mg protein

Question 12

How is yield calculated?

Answer 12

total Activity before purification/total activity before purification; ideally we want it to stay the same but in reality we will lose some of our targeted protein

Question 13

How is purity calculated?

Answer 13

S.A. after/S.A before; we want this to go up because it means we have more of our target protein per all protein in the sample

Question 14

Order of specificity for chromatography

Answer 14

Affinity (most, but most expensive), Ion exchange, size exclusion (least, easiest to do)

Question 15

What are the ingredients to make the 5x buffer for SDS-Page?

Answer 15

10% SDS, 20% glycerol, 0.2M Tris HCl (pH 6.8); 0.05% bromophenol blue; 10mM beta-mercaptoethanol

Question 16

Briefly describe the SDS-Page procedure

Answer 16

Protein samples are boiled for 5 min in 5x loading buffer; Gel is cast and poured between glass or plastic plates; combs are used to form loading wells; gel is placed vertically in electrophoresis apparatus where top and bottom tanks are filled with buffer; samples and ladder are loaded into the wells, apparatus is turned on to 250V and runs till blue dye reaches the bottom; once finished, gel is removed and immersed in: coomassie blue for 10 min, then buffer for 10 min (repeat 2-3x); OR silver nitrate for more sensitive applications (,pre complicated and time consuming) OR fluorescent dye staining (sensitive, but requires specialized equipment)

Question 17

what purpose does glycerol serve in the buffer?

Answer 17

to increase density and viscosity so protein can sink to the bottom and separate

Question 18

the dye must be the same charge as the SDS (negative) so it runs in the same direction

Answer 18

ye

Question 19

i think coomassie blue is the least sensitive protein staining method we studied but idk

Answer 19

ye

Question 20

What is the purpose of a stacking gel?

Answer 20

To help increase band resolution

Question 21

What composes a stacking gel?

Answer 21

The top gel (stacking gel) made up of 5% acrylamide, pH 6.8 with Tris-HCl; Bottom gel (running gel) with ~12% acrylamide, pH 8.8 w/ Tris HCl; upper buffer: pH 8.3 w glycine, pI=6

Question 22

Why would some parts of standard curve not be linear?

Answer 22

The proteins travelling may be too big or too small to be effectively separated by the acrylamide gel at that specific concentration

Question 23

What is the horizontal plane of a 2D protein gel?

Answer 23

size (also called dimension 2)

Question 24

What is the vertical plane of a 2D protein gel?

Answer 24

charge (also called dimension 1)