Kap 6: Electrophoretic techniques

Question 1

There are two types of gel units for electrophoresis which two?

Answer 1

vertical slab gel

horizontal gel

Question 2

Why is it essential to use a buffer when electrophoresis is carried out?

Answer 2

To maintain a constant state of ionization of the molecules being separated. Any variation in pH would therefore alter the overall charge –> mobilities (rate of migration in the applied field)

Question 3

What does the voltage (potential difference) generate?

Answer 3

a potential gradient

Question 4

By knowing the potential gradient and molecule net charge one can calulate ?

what tries to oppose the force?

Answer 4

the force of migration

the frictional resistance which retards the molecules baring charge

Question 5

the frictional resistance depend on 4 things, which?

Answer 5

hydrodynamic size, shape, pore size of medium and buffer viscocity.

Question 6

What is electrophoretic mobility?

what does it tell us when an potential difference is applied?

Answer 6

the ratio of the velocity of the ion to field strength

that molecules with different overall charges will begin to separate owing to their different electrophoretic mobilities. Even molecules with similar charges will begin to separate if they have different molecular sizes, since they will experience different frictional forces

Question 7

What justifies that electrophoresis is an incomplete form of electrolysis?

Answer 7

that the electric field has to be removed before the molecules in the sample reaches the electrodes.

Question 8

What does mainly conduct the current in the solution?

while a small proportion being conducted by the sample ions.

Answer 8

buffer,

Question 9

Heating of the electrophoretic medium has the

following effects:

Answer 9

a) An increased rate of diffusion of sample and buffer
ions –> broadening of the separated samples.
b) formation of convection currents. -> mixing of
separated samples
c) Thermal instability of samples that are rather sensitive
to heat
d) A decrease of buffer viscosity –> resistance reduction
for medium

Question 10

If a constant voltage is applied, the current increases during electrophoresis owing to the ____ in resistance, and even if the resistance is decreased the increased current increases the heat output still

Thereby one would use stabilized power supply, which provides constant power and thus eliminates fluctuations
in heating –> but this give us ___ heat generation which is a problem

this in turn means that for slab gels although heat is generated uniformly through the medium, heat is removed only from the ___

Answer 10

decrease
constant
edges, where we have less viscosity thus less electrophoretic mobility

Question 11

What give rise to electroendosmosis which affects electrophoretic separation?

Answer 11

presence of charged groups on the surface of the support medium can get ionized at a pH, therby creating a electrical double layer or region of charge seperation

When a voltage is applied, cations in the electrolyte
near the capillary wall migrate towards the cathode, pulling electrolyte solution with them. This creates a net electroendosmotic fl ow towards the cathode.

Question 12

Which two factors does a support medium cut down?

Answer 12

diffusion and convection currents

Question 13

6.2 SUPPORT MEDIA AND BUFFERS
6.2.1 Agarose Gels
a) Agarose is a linear polysaccharide made up of the
basic repeat units of: ___
b) Which concentration of agarose is used typiically?
c) The gelling properties are attributed to: ___
d) the gel is a cross-linked structure what property
does this give the gel?
e) what controlls the pore size in the gel?
f) Although the gel is free of charge, sugar residue
substitutions occur to varying degrees –>
electroendosomosis and ionic interactions, so what
is the outcome of this varying degrees of
substitutions ?
g) Does lower sulphate content in the agarose give
lower purity?
h) Agarose gels are used for the electrophoresis of
both proteins and nucleic acids. For proteins, the
pore sizes of a 1% agarose gel are large relative to
the sizes of proteins, so becuase the pore size and
molecule size are more comparable we get ___
effects

Answer 13

a) agarobiose
b) 1-3 %
c) both inter- and intramolecular hydrogen bonding within
and between the long agarose chains
d) good anti-convectional properties
e) the initial concentration of agarose
f) Agarose is sold with different purity grades, based on
the sulfate concentration
g) no
h) fractional effects

Question 14

6. 2 SUPPORT MEDIA AND BUFFERS
   
   6. 2.1 Agarose Gels

give an advantage in regard to temperature!

Answer 14

becuase agarose has a low melting temperature thus the gel can be reliquified, so DNA samples seperated in the gel can be cut put of the gel, returned to solution and be recovered.

Question 15

Horizontal slab gels are invariably used for ____ focussing or ___-electrophoresis in agarose

Answer 15

isoelectric

immuno

Question 16

Horizontal gels are also used routinely for ___

and ___ gels

Answer 16

DNA

RNA

Question 17

What is the most common buffer for seperating DNA?

is it a strong buffer?

Answer 17

TRIS-acetate; containing EDTA ( TAE) with a typical pH of 8.3

no

Question 18

What is the most common buffer for seperating RNA?

Answer 18

TRIS-borate with EDTA (TBE)

Question 19

Regarding the most common buffer for seperating RNA, what is an disadvantage of borate?

Answer 19

it acts as an inhibitor for many enzymes

Question 20

Between TAE and TBE, which gives superior separation of smaller fragments (< 2kb)?

Answer 20

TBE

Question 21

Electrophoresis in acrylamide gels is frequently referred to as ___

Answer 21

PAGE

Question 22

What compound makes the polyacrylamide cross-linked?

what does it introduce?

in order to get polyacrylamide chains it is via polymerization of acrylamide monomers through a free-radical catalysis, but what initiates this process?

Answer 22

bis-acrylamide

2nd site for extension

ammonium persulfate and TEMED

Question 23

What does TEMED do in the procees of formation of bis-polyacrylamide gels

Answer 23

catalyses the decomposition of the persulfate ion to give a free radical that polymerasises with an acrylamide monomer –> thus generating long chains of acrylamide that occasionaly bis-acrylamide is introduced

Question 24

The degassing of the gel solution has two purposes!

Answer 24

1. to negate possible oxygens from reacting with remaining free radicals
2. becuase the polymerization is an exothermic reaction, the warming up of the solution liberates air bubbles, which is prevented.

Question 25

Acrylamide gels are defined in terms of the total percentage of acrylamide present, and the __ __ in the gel can be varied by changing the concentrations of
both acrylamide and __-___

Answer 25

pore size

bis-acrylamide

Question 26

Does low percentage acrylamide gels have large pore sizes?

what are these used for?

Answer 26

yes

electrophoresis of proteins, where free movement
of the proteins by electrophoresis is required without any noticeable frictional effect, for example in:
* flat-bed isoelectric focussing ( Section 6.3.4 )
* the stacking gel system of an SDS–polyacrylamide gel
(10 - 20% –> sieving effect)

but also for DNA

Question 27

6. 3 ELECTROPHORESIS OF PROTEINS
7. 3.1 SDS-PAGE

because the method is based on the separation of proteins according to size, it can also be used to determine the:

Why does SDS binds strongly to the thermally denatured
protein?

What is the purpose of beta-Mercaptoethanol or DTT?

Answer 27

relative molecular mass of proteins

becuase it’s a anionic detergent

to break disulfide briges that are holding together the protein teriary structure.

Question 28

In SDS page why would one add sucrose or glycerol?

Answer 28

to give sample densiy

Question 29

In conventional discontinuous gel electrophoresis why do we have a stacking gel?

how is this achieved?

Answer 29

to concentrate the protein sample into a sharp band before it enters the main separating gel

by utilising differences in ionic strength and
pH between the electrophoresis buffer (pH 8.8) and the stacking gel buffer (pH 6.8) and
involves a phenomenon known as isotachophoresis

Question 30

The negatively charged protein–SDS complexes now
continue to move towards the anode, and, because they have the same charge per unit length, they travel into the separating gel under the applied electric fi eld with the same ___. However, as they pass through the separating gel the proteins separate, owing
to the molecular ___ properties of the gel

Answer 30

mobility

sieving

Question 31

What is the advantage of single gel electrophoresis

Answer 31

in the preparation we dont use SDS, so the prepared gel can be used either as SSDS, SDS/denaturing or native. gel

Question 32

For PAGE how do we keep biological activity of a particluar enxyme?

Since all the proteins in the sample being analysed carry their ___ ___ at the pH of
the gel (normally pH 8.7), proteins separate according to their different electrophoretic
mobilities and the ___ effects of the gel

Answer 32

by not using SDS, thus we need a non-denaturing condition

native charge

sieving

Question 33

What are the two advantages with gradient gels?

Answer 33

1. gives a much greater range of protein molecular
   weights can be separated than on a fixed-percentage
   gel.
2. proteins with very similar Mr values may be resolved
   due to the pore size limit for each protein

Question 34

6.3.4 Isoelectric Focusing Gels

for which type of substances is this method ideal for?

Separation is achieved by applying a potential difference across a gel that contains a pH gradient, how is this formed?

Since this method requires the proteins to move freely according to their charge under the electric field, IEF is carried out in low-percentage gels to avoid any ___ within the gel

Why would one use agarose in IEF?

proteins that are initially at a pH region below their isoelectric point will be positively charged and
will initially migrate towards the ___. As they proceed, however, the surrounding
pH will be steadily increasing down the tube, and therefore the positive charge on the protein will
decrease correspondingly until eventually the protein arrives at a point where the pH
is equal to its isoelectric point. The protein will now be in the ___ form with no
net charge, so further movement will cease

after the electrophoresis why cant we directly dye the gel?

Answer 34

amphoteric, such as proteins

with ampholytes, that should have a pH range covering pI of sample

sieving

for proteins having high Mr we need gels that can undergo sieving, even in low-percentage acrylamide gel

cathode

zwitterion

because the ampholytes will stain too, giving a totally blue gel. The gel is, therefore:
1. first washed with fixing solution (e.g. 10% (v/v)
trichloroacetic acid).
2. This precipitates the proteins in the gel and allows the
much smaller ampholytes to be washed out.
3. The gel is stained with Coomassie Brilliant Blue and
4. then destained ( Section 6.3.7 ).

Question 35

Because IEF is a highly sensitive analytical technique, it is particulary useful for studying _____ in a protein

Answer 35

microheterogeneity (for example different phosphorylated forms) which cant be observed with SDS gels

Question 36

For IEF the sudy of microheterogeneity is particulary useful for separating ____

how can these enzyes be detected?

Answer 36

isoenzymes

in the
gel either by washing the unfi xed and unstained gel in an appropriate substrate or
by overlayering with agarose containing the substrate

Question 37

2D SDS-PAGE
is a combination of:

what does the gel contains?

The denatured proteins therefore separate in
this gel according to:

Answer 37

1D IEF + SDS-PAGE

contains ampholytes (for forming the pH gradient) together with 8 M urea and a non-ionic detergent, both of which denature and maintain the solubility of the proteins being analysed

their isoelectric points before the SDS PAGE region

Question 38

Name one protein staining?

what mixture is it carried in?

Answer 38

CBB

0.1% (w/v) CBB + methanol:water:glacial acetic acid (45:45:10, by volume)

Question 39

The CBB stain is not used for staining cellulose acetate (or indeed protein blots) becuase?

so how it it possible?

Answer 39

proteins bind quite strongly to the paper

1. proteins first denatured in 10% (v/v) trichloroacetic acid
2. immersed in solution of a dye that does not stain the
   support material

Question 40

After a PAGE we achieve fractionation of a protein mixture during the electrophoresis process how can we further investigate the fractionations?

how?

Answer 40

with a western blot

1. transfer protein from gel to cellulose paper or PVDF via
   electroblotting process
2. probing the blot; antibody, but first incubate blot in a
   protein solution - in order to block remaining
   hydrophobic binding sites on the cellulose sheet
3. then incubation in a dilution of an antiserum (primary
   antibody) that will bind to antibody
4. in order to visualise this binding the blot is then
   incubated firther in a solution of a 2nd antibody
   (labeled- enzyme linked) - usually a alkaline
   phosphatase to find the antiserum and converts the
   substrate into an insoluble coloured product that is
   precipitated onto the nitrocellulose
5. By careful comparisons of the blot with a stained gel of
   the same sample, the protein of interest can be identifi
   ed

Question 41

6.4.1 Agarose Gel Electrophoresis of DNA

For the majority of DNA samples, electrophoretic separation is carried out in ___ gels. This is because most DNA molecules and their fragments that are analysed routinely are considerably larger than proteins

Answer 41

agarose

Question 42

6.4.1 Agarose Gel Electrophoresis of DNA

Rather than using Mr for DNA one typically refer the size in bp (kbp), where 1 kb is Mr: ____

Answer 42

620 000

Question 43

6.4.1 Agarose Gel Electrophoresis of DNA

What is the reason for seperation in agarose gel?

Answer 43

the DNA samples should move towards the anode with the samemobility under an applied electrical field but becuase they migrate on the gel matrix they seperate due to movement resistance due to the length of the DNA rods

therefore the mobility of DNA molecules during
gel electrophoresis will depend on the size.

Question 44

6.4.1 Agarose Gel Electrophoresis of DNA
Why is no stacking gel needed?

a voltage gradient of about 1.5Vcm −1 overnight. A higher
voltage would cause excessive ____

After DNA in the gel is stained at which wavelength can we visualise the result

What does ehtinium bromide or SYBR green do as a staining agent for DNA?

Answer 44

because the mobilities of DNA molecules are much greater in the well than in the gel, and therefore all the molecules in the well pile up against the gel within a few minutes of the current being turned on, forming a tight band at the start of the run.

heating

300 nm

it interchelates by binding between the stacked base-pairs of DNA.

Question 45

6.4.2 DNA Sequencing Gels
why is polyacrylamide gels used instead of agarose?

The DNA bands of interest can be cut out of the gel and the DNA recovered by ___ then the DNA can be recovered by ____ of the supernatant with ethanol

Answer 45

becuase in order to sequence DNA we cant be confined to electrophoresis for relatively small DNA molecules we need a small-pored gel to better separate a known sample of DNA which is needed becuase we use long gels to maximize separation.

Therfore electrophoresis in agarose can be used as a preparative method for DNA to separate big fragments from small ones.

centrifugation

precipitation