Electrophoresis

Question 1

Definition

Answer 1

Separate biological molecules based on size, shape + charge (E. g DNA, Proteins)

Question 2

How does it work?

Answer 2

Electrical current applied to mixture move towards opposite charge at different speeds

Question 3

1D or 2D

Answer 3

1 plane of separation or 2.

Question 4

Densitometry

Answer 4

Light passed through gel + transmittance measured. Computer calculates amount of molecule present. Semi- quantitative

Question 5

Applications

Answer 5

Diagnostic, Forensic (DNA fingerprint), Paternity

Question 6

Samples

Answer 6

Blood, urine,FNAC, Homogenates from biopsy

Question 7

Plasma Proteins

Answer 7

Specific set of functions + easy to assay = diagnostic tool and monitoring clinical progress

Question 8

Cellulose acetate electrophoresis ( CAE) method + application

Answer 8

Sample at cathode end, pH at 8.6 = negative charged proteins, Move to anode and visualized. used for haemoglobin in sickle cell

Question 9

Electrophoretic Graph

Answer 9

4 major bands : Albumin, Alpha 1, Alpha 2, Beta 1+2, Gamma

Question 10

PAGE : 2 types

Answer 10

Denaturing= Separation based on size
Non denaturing (native)= Separation on mass: charge ratio

Question 11

Native PAGE benefits + separation

Answer 11

Retain structure + enzymatic activity
Migration on size, shape + charge

Question 12

SDS- PAGE (charge+ uses)

Answer 12

Gives proteins negative charge + denatures it
Estimate purity+ molecular weight
Separation by size

Question 13

PAGE % acrylamide

Answer 13

Low = Bigger pores , High: smaller

Question 14

Western Blotting Method

Answer 14

Proteins separated by SDS-PAGE, transferred to membrane, Ab added + binds specific protein, Incubation with secondary Ab-enzyme conjugate, Incubated with substrate = product

Question 15

Zymography (uses + method)

Answer 15

Measures enzymatic activity
substrate bound to gel
Breakdown of substrate= clear band

Question 16

Agarose Gel Electrophoresis Principle

Answer 16

separate + analyse DNA + RNA based on molecular weights

Question 17

Agarose Gel Electrophoresis visualization

Answer 17

Ethidium Bromide staining = fluorescence under UV

Question 18

Isoelectric Point / focussing

Answer 18

Proteins separated in PH gradient. Stop migrating when pI reached
pI = Zero charge
low pH= Positive
high pH = negative

Question 19

2D electrophoresis

Answer 19

1D= separation by pI
2 D= separation by MW ( SDS- PAGE)
Identify proteins using Mass Spec