PAGE

Question 1

What is page

Answer 1

Polyacrylamide gel electrophoresis

Question 2

What is electrophoresis

Answer 2

Movement of charged particles under the influence of uniform electric field

Question 3

Particles sort acc to?

Answer 3

Size
Charge

Question 4

Electrophoresis components

Answer 4

Current
Gel (sieve)
Buffer

Question 5

Is polyacrylamide gel vertical or horizontal

Answer 5

Vertical

Question 6

Structure of acrylamide Bis polymerization

Answer 6

2 chains of acrylamide linked by bis acrylamide

Question 7

The polymerization process

Answer 7

Initiated by APS
catalyzed by TEMED

Question 8

Pore size in the gel is determined by

Answer 8

Ratio of acrylamide to bis acrylamide and concentration of acrylamide.

Question 9

Gel percentage is

Answer 9

Where the desired protein when migrate till the second half of the gel

Question 10

Optimum separataion

Answer 10

2nd half of gel

Question 11

Gel layers

Answer 11

Stacking> pH 6.8>low ionic strength , large pores
Running> pH 8.8> high ionic strength, small pores

Question 12

Buffers

Answer 12

Running>conductive medium so electric charge pass (Tris and glycerine)
Sample>carrying the sample, conductive(Tris,glycerol, bromophenol blue)

Question 13

Why bromophenol blue

Answer 13

Tracking dye

Question 14

Why glycerol

Answer 14

To weigh down the samples into the well and prevent them from flowing to neighbouring wells

Question 15

Gel preparation steps

Answer 15

All components except APS and TEMED
At last as the polymerisation is fast

Question 16

Continuous vs discontinuous buffer systems

Answer 16

Continuous> same buffer in all gel, uniform separating matrix, fuzzy and unresolved protein bands (nucleic acid analysis)
Discontinuous> different buffers, 2 sections, improved resolution,

Question 17

Molecules move from

Answer 17

Cathode to anode

Question 18

Native PAGE

Answer 18

Non denaturing
Charge and size

Question 19

Native PAGE applications

Answer 19

Enzymatic activity
Binding interactions
Detection of cofactors

Question 20

SDS PAGE

Answer 20

denaturing
Acc to size only

Question 21

Why b- marcaptoethanol in buffer

Answer 21

As a reducing agent to break disulfide bonds

Question 22

Denaturing PAGE has…buffers

Answer 22

Running and sample
Discontinuous system

Question 23

What id SDS
Function

Answer 23

sodium dodecyl sulphate
A detergent with hydrophobic tail and -vely charged sulfate group, maintain primary structure only and coats the protein with negative charge

Question 24

Why glycine

Answer 24

Amino acid affected by Ph, aligns proteins in stacking gel

Question 25

SDS PAGE applications

Answer 25

Purity assessment
protein expression
Western blotting

Question 26

Coomaise blue staining

Answer 26

Wash-stain-destain-observe

Question 27

Smeared gel reasons

Answer 27

Contamination
Insufficient heating so proteins are not completely denatured
Gel overloading