SDS-PAGE

Question 1

SDS

Answer 1

Detergent
Sodium dodecyl sulfate
Denatures proteins when boiled with reducing agents (2-me or DTT)
Binds to prot and denatures so they assume random coil configuration
Adds neg charge

Question 2

Reducing agent

Answer 2

2-mercaptoethanol (2-me)
dithiothreitol (DTT)
breaks disulfide bonds to -SH groups allowing subunits (H and L chains) to separate
Necessary for multi-chain samples

Question 3

Charge-to-mass ratio

Answer 3

Amt SDS/unit weight is constant for all prot
Neg charge from sulfate groups > intrinsic charge
Uniform

Question 4

Protein mobility

Answer 4

Inversely proportional to log of mw

Question 5

Porosity of gel

Answer 5

Inversely proportional to % acrylamide used to make gel

Question 6

Laemmli method

Answer 6

Discontinuous buffer system
Improves resolution
Highly porous pH 6.8 on top
Less porous pH 8.8 on bottom

Question 7

Glycerol

Answer 7

Added to sample buffer to incr density so samples will sink in wells
Sucrose can also be added

Question 8

Bromophenol blue

Answer 8

Added to samples
Easily visible
Dye front
7kDa

Question 9

APS

Answer 9

Ammonium persulfate
Produces free radicals
Catalyze polymerization

Question 10

TEMED

Answer 10

Smells like ass

Accelerates rate of formation of free radicals from APS

Question 11

Acrylamide

Answer 11

Unpolymerized is neurotoxin
Bis-acrylamide provides crosslinking
ratio of 37.5:1 –> determines porosity

Question 12

Separating gel

Answer 12

Less porous
pH 8.8
12% acrylamide
Higher (1.5M) salt concentration
Higher pH favours ionization of glycine so it can travel immediately behind Cl- at edge of moving boundary
Zone is now of uniform voltage and pH and prot can be separated by size

Question 13

Stacking gel

Answer 13

More porous
pH 6.8
4% acrylamide
Lower (0.5M) salt concentration
Creates high voltage gradient
Prot move quickly
Concentrates prot into tighter, narrower bands
Cl- forms edge of moving boundary
Glycine forms trailing edge
B/w edges is area of lower conductivity and steeper voltage gradient which sweeps prot from sample and deposits them on gel

Question 14

Voltage

Answer 14

100V through stacking gel to prevent warping/smiling

200V through separating gel

Question 15

Gel porosity

Answer 15

Concentration of monomer acrylamide
Amt bis-acrylamide
Polymerization conditions (buffer, TEMED amt)

Question 16

Boiling

Answer 16

Breaks down secondary structure

Removes prot from beads

Question 17

Lc mw

Answer 17

23-25kDa

Question 18

Hc mw

Answer 18

50-55kDa

Question 19

Ab mw

Answer 19

150-160kDa

Question 20

MW markers

Answer 20

Measure migration distance of known prot standards to create standard curve

Question 21

Sample Buffer

Answer 21

Contains SDS to coat prot, reducing agent, sucrose/glycerol and bromophenol blue

Question 22

Running Buffer

Answer 22

Allows for recirculation of charge

Contains Tris-HCL and Glycine to help in transfer of charge