UNIT VI
Electrophoresis of proteins
1.Electrophoresis was first developed byArne Tiselius.
2. Charged particles experience force when they are placed in Electric field
3. The force experienced by a particle of charge Q when placed in electric field E is given by
Q.E
4. The acceleration of charged particle caused by electrophoretic attraction is opposed by
(i) Friction (ii) electrophoretic retardation (iii) relaxation effect (i), (ii) and (iii)
5. The units of electophoretic mobility is meter2/volts.
6. Electrophoresis is used to separate molecules based on charge
7. At low pH values, alpha amino group will be in ____________ form.NH3+.
8. At high pH values, alpha amino group will be in ____________ form.COO-9S. For a protein at isoelectric point, electrophoretic mobility is Zero
10. since the ___________ groups in polynucleotides are very negatively charged, they generally move towardsanode.Phosphate
Electrophoresis of nucleic acids
1. Friction force which decelerates a charged particle in electrophoresis is given by F.U
2. For spherical particle at very low reynolds number, friction coefficient (f) viscousity(n) and
radius of particle(R) are related by the equation f=6 PnR
3. The electrophoretic mobility(m) is related to electron charge (e), valence (Z), viscosity(n) and radius of particle(R) by the equation n=(Ze)/(6 PnR).
4. The combination of negative and positive charge around a charged particle to have electroneutrality is called double layer.
5. The value of potential at the at the shear boundary of a charged colloidal particle is called
Zeta potential
6. The electric potential of a charged colloid whose electric potential at the surface is So drops to zero at infinite distance according to the relation (here S is potential at distance r, K is debye huckel constant and R is the radius of the particle S=So exp(-K(r-R))
7. The units of Debye Huckel constant are C2/Jm
8. Electrophoretic mobility _________ as size of ion __________decreases, increases
9. The distorted double layer exerts a force on particle known as Relaxation effect.
10. Huckel’s equation relates electrophoretic retardation force(F) and dielectric constant(P), zeta potential(z) charge(Q), electric field(E), permittivity(Po) and radius of particle(R) by the equation F=[4 P PoPzR-Q]E
One dimensional and two dimensional gels.
1. Which of the following method is used to recover the separated sample in electrophoresis?
(i) blotting onto paper (ii) eluting electrophoretically with flow (iii) scrapping some of the gel into beaker (i), (ii) and (iii)
2. The stabilizing media used for electrophoresis must have which of the following properties?
It should be inert.
3. Which of the following properties should a stabilizing media used for electrophoresis have?
The media must not have any residual charges.
4. In _______ method, electrophoresis is conducted in a gel slab with a gradient in pore size.
Pore limit electrophoresis
5. In SDS-PAGE, protein solution is treated with SDS and mercaptoethanol to Destroy the disulfide bond.
6. Agarose is a polymer of D-Galactose and 3,6-Anhydrogalactose.
7. IN agarose gels, the polymeric chains are held together by H-bonds
8. Occurrence of electroosmosis in agarose gels can be minimized by Alkali pre-pretreatment.
9. ____________ was the first gel to be used for electrophoresis Starch.
10. Filter paper was the first anti convective medium used for zone electrophoresis
11. The maximum number of compounds(n2d) that can be separated by 2-D electrophoresis is related to the number of compounds that are separated by single independent separations(n1 and n2) by the equation n2d=n1.n2.
12. The sequential 2-D electrophoresis separation by doing iso-electric focusing first followed by SDS-PAGE is a _____________ method.Completely independent.
13. In separation by iso electric focusing followed by SDS-PAGE the molecules are separated
First based on iso-electric point and then on molecular size.
14. _________ electrophoresis is used for separating Yeast chromosomes.Pulsed gel.
15. In a continuous 2-D electrophoresis, the net velocity is given by
Vector addition of horizontal and vertical velocity.
16. In continuous 2-D electrophoresis, the retention time (T), length of the gel(L), and velocity of the buffer(Vb) is related by the equationT=L/Vb.
17. The angle(A) between electrophoretic migration velocity(u) and the buffer velocity(v) in continuous 2-D electrophoresis is given by tan(A)= u/v.
18. scale up of electrophoresis is difficult due to which of the following reasons?
(i)It is difficult to control convection (ii) The resolving power is low at modest voltages (iii) cooling is difficult(i),(ii) and (iii).
19. Which of the following methods can be used to prevent thermal convection
adding a density gradient.
20. Electric field(E), electric potential(V) and length (L) are related by the equationE=V/L
Capillary and pulse field electrophoresis
1. What is the inside diameter of the capillary used for capillary electrophoresis.
a. 80-100 micrometer
b. 25-75 micrometer.
c. 10-25 micrometer
d. 100-110 micrometer ans b
2. Which among the following is the advantage of using narrow capillary for electrophoresis
(i) the cooling is efficient (ii) small rayleigh number (iii) convective motion is inhibited.
a. (i) and (ii)
b. (ii) and (iii)
c. (i), (ii) and (iii)
d. (i) and(iii) ans c
3. Why are long capillaries used when very high voltages are used for electrophoresis?
a. To dissipate joule heating.
b. To facilitate better separation
c. To obtain higher temperatures
d. To slow down the separation process ans a
4. Capillary electrophoresis is not preferred to separate proteins due to which of the following reasons?
(i) proteins adsorb to the walls of capillary (ii) high temperatures denature proteins (iii) resolution is poor.
a. Only(i).
b. (i) and (ii)
c. (i) and (iii)
d. (i), (ii) and(iii) ans a
5. Protein adsorption on capillary wall can be controlled by operating under __________.
a. Acidic conditions
b. Basic conditions
c. Neutral conditions
d. Low concentration of salts in buffer ans b
6. Which among the following is an example of free solution electrophoresis.
a. Moving boundary electrophoresis
b. Capillary electrophoresis
c. Immuno electrophoresis
d. Affinity electrophoresis ans a
7. In free solution electrophoresis, thin chambers are used for which of the following reasons
(i) To prevent convection (ii) to have low rayleigh number (iii) for efficient cooling
a. (i) and(ii)
b. (ii) and(iii)
c. (i), (ii) and (iii)
d. only (iii)
8. ________________ method does electrophoresis of colloidal particles between two slides
a. microscope electrophoresis.
b. capillary electrophoresis
c. affinity electrophoresis
d. immuno electrophoresis ans a
9. The gel layer is kept thin
a. to have better heat dissipation.
b. to facilitate convection
c. to have high rayleigh number
d. for decreased mechanical strength ans a
10. A thin gel is used for electrophoresis
a. to have high mechanical strength.
b. to reduce heat dissipation
c. better resolution
d. to facilitate convection. Ans a
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