Supplementary practical - SDS-PAGE

Question 1

on the polyacrylamide gel, mobility of molecules is directly proportional to what?

Answer 1

log10 of the mass of the protein

heavier proteins will be closer to the loading well - lighter ones move further down the gel

Question 2

which specific cellular compartment was targeted by the increased expression of protein via the insertion of a novel gene?

Answer 2

periplasm

Question 3

what does the beer-lambert law describe with regards to path length, conc and absorbance

what shape will this law take on a graph? which coefficient is the gradient

Answer 3

as the path length or conc increases, the absorption increases

straight line, extincion coefficient is gradient

Question 4

the unknown goes on the x axis. what is the unknown for this practical?

Answer 4

protein concentration

Question 5

what does a bradford assay measure? is it colorimetric?

Answer 5

the presence of basic amino acids (his, lys, arg), which form a protein-dye complex

yes it is colorimetric

Question 6

which compound binds to the lac repressor, releasing it, switching on T7, causing transcription of proteins?

Answer 6

IPTG

Question 7

what is the promoter of PET21a called? is PET21a a lac repressor or promoter?

Answer 7

T7

repressor

Question 8

what is the pH of the upper stacking gel in SDS PAGE?

does it have small or large pores?

does it have a high or low percentage of acrylamide?

Answer 8

6.8

large pores due to low % acrylamide

Question 9

what is the pH of the lower resolving/main gel in SDS PAGE?

does it have small or large pores?

does it have a high or low percentage of acrylamide?

Answer 9

8.8

small pores due to high % acrylamide

Question 10

what are the main carriers of the current in the gel in SDS PAGE?

Answer 10

proteins with SDS

Question 11

what is the mobile anion in both gels of SDS PAGE? which molecule has no charge in the upper stacking gel?

Answer 11

CL-

glycine

Question 12

what is the function of Tris buffer?

Answer 12

keep pH constant

Question 13

what is the function of glycerol in this practical?

Answer 13

provides weight to solution so material sits at bottom of wells

Question 14

what is SDS? what does it do to cells and proteins? how?

what does it add specifically to proteins?

Answer 14

a detergent which lyses cells and solubilises/denatures the proteins by breaking S=S bonds

also adds a negative charge to proteins so it runs down the gel to anode

Question 15

which reagent gives colour to the reaction so you can see how the gel is running?

Answer 15

bromophenol blue

Question 16

how do you calculate retardation factor? which axis would it be plotted on against log10 MW?

Answer 16

distance moved by protein divided by distance moved by dye front

plotted on x axis

Question 17

what can IPTG, mimicing allolactose, induce, when cells are provided with lactose?

Answer 17

the lac operon

Question 18

transcription of the lac structural genes only occurs when ……. is present and ……. is absent

Answer 18

lactose present
glucose absent

Question 19

what does the lac repressor bind to?

Answer 19

the lac operator

Question 20

what does SDS coat denatured proteins evenly in?

Answer 20

negative charge

Question 21

after proteins have been denatured and coated evenly in negative charge by SDS, what does this prevent them from doing?

Answer 21

folding

Question 22

on an acrylamide gel, how would you measure the distance migrated by an unknown protein?

Answer 22

measure from the TOP of the gel down to the middle of the unknown protein band. Then you would measure the unknown band against the nearest MW standard.

Question 23

on an acrylamide gel, how would you measure the distance migrated by the dye front?

Answer 23

using a ruler you would measure from the dye front’s starting line at the bottom of the gel, up to the top of the gel where it has stopped moving. measure in mm

Question 24

how would you calculate the Rf value of an unknown protein you have on your gel?

Answer 24

distance moved by protein (mm) divided by the distance moved by the dye front (mm)

Question 25

how would you estimate the MW of an unknown protein from a drawn standard curve (log MW against Rf value)?

it has an Rf value of 0.55

Answer 25

on the x axis (Rf value), find 0.55.

plot a line up to the standard curve, then plot another line across to the y axis to read off the log MW value

then to de log the value, you would do 10^ of whatever the log MW value is

Question 26

a standard curve of logMW (y axis) against Rf value (x axis) should have which shape? which direction should it be going?

Answer 26

it is a straight line coming from top left to bottom right of graph

Question 27

what is the ideal voltage to run your SDS-PAGE electrophoresis at?

Answer 27

40-180 V

Question 28

how should the gel be trimmed for analysis after the dyes have run? what shape should it have?

Answer 28

should be rectangular in shape

should be trimmed so that all bands are contained, but none of the wells (cut off the wells)

Question 29

when staining the gel, how many rounds of staining are done, followed by how many rounds of de-staining?

Answer 29

one round of staining followed by 2 rounds of destaining

Question 30

when plotting. standard curve for Rf values and logMW values, what would you put on the x and then the y axis?

Answer 30

x axis = Rf values (calculated from gel)
y axis = logMW values