Kap 6: Electrophoretic techniques Flashcards
There are two types of gel units for electrophoresis which two?
vertical slab gel
horizontal gel
Why is it essential to use a buffer when electrophoresis is carried out?
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)
What does the voltage (potential difference) generate?
a potential gradient
By knowing the potential gradient and molecule net charge one can calulate ?
what tries to oppose the force?
the force of migration
the frictional resistance which retards the molecules baring charge
the frictional resistance depend on 4 things, which?
hydrodynamic size, shape, pore size of medium and buffer viscocity.
What is electrophoretic mobility?
what does it tell us when an potential difference is applied?
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
What justifies that electrophoresis is an incomplete form of electrolysis?
that the electric field has to be removed before the molecules in the sample reaches the electrodes.
What does mainly conduct the current in the solution?
while a small proportion being conducted by the sample ions.
buffer,
Heating of the electrophoretic medium has the
following effects:
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
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 ___
decrease
constant
edges, where we have less viscosity thus less electrophoretic mobility
What give rise to electroendosmosis which affects electrophoretic separation?
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.
Which two factors does a support medium cut down?
diffusion and convection currents
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
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
- 2 SUPPORT MEDIA AND BUFFERS
- 2.1 Agarose Gels
give an advantage in regard to temperature!
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.
Horizontal slab gels are invariably used for ____ focussing or ___-electrophoresis in agarose
isoelectric
immuno
Horizontal gels are also used routinely for ___
and ___ gels
DNA
RNA
What is the most common buffer for seperating DNA?
is it a strong buffer?
TRIS-acetate; containing EDTA ( TAE) with a typical pH of 8.3
no
What is the most common buffer for seperating RNA?
TRIS-borate with EDTA (TBE)
Regarding the most common buffer for seperating RNA, what is an disadvantage of borate?
it acts as an inhibitor for many enzymes
Between TAE and TBE, which gives superior separation of smaller fragments (< 2kb)?
TBE
Electrophoresis in acrylamide gels is frequently referred to as ___
PAGE
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?
bis-acrylamide
2nd site for extension
ammonium persulfate and TEMED
What does TEMED do in the procees of formation of bis-polyacrylamide gels
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
The degassing of the gel solution has two purposes!
- to negate possible oxygens from reacting with remaining free radicals
- becuase the polymerization is an exothermic reaction, the warming up of the solution liberates air bubbles, which is prevented.
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 __-___
pore size
bis-acrylamide
Does low percentage acrylamide gels have large pore sizes?
what are these used for?
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
- 3 ELECTROPHORESIS OF PROTEINS
- 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?
relative molecular mass of proteins
becuase it’s a anionic detergent
to break disulfide briges that are holding together the protein teriary structure.
In SDS page why would one add sucrose or glycerol?
to give sample densiy
In conventional discontinuous gel electrophoresis why do we have a stacking gel?
how is this achieved?
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
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
mobility
sieving
What is the advantage of single gel electrophoresis
in the preparation we dont use SDS, so the prepared gel can be used either as SSDS, SDS/denaturing or native. gel
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
by not using SDS, thus we need a non-denaturing condition
native charge
sieving
What are the two advantages with gradient gels?
- gives a much greater range of protein molecular
weights can be separated than on a fixed-percentage
gel. - proteins with very similar Mr values may be resolved
due to the pore size limit for each protein
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?
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 ).
Because IEF is a highly sensitive analytical technique, it is particulary useful for studying _____ in a protein
microheterogeneity (for example different phosphorylated forms) which cant be observed with SDS gels
For IEF the sudy of microheterogeneity is particulary useful for separating ____
how can these enzyes be detected?
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
2D SDS-PAGE
is a combination of:
what does the gel contains?
The denatured proteins therefore separate in
this gel according to:
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
Name one protein staining?
what mixture is it carried in?
CBB
0.1% (w/v) CBB + methanol:water:glacial acetic acid (45:45:10, by volume)
The CBB stain is not used for staining cellulose acetate (or indeed protein blots) becuase?
so how it it possible?
proteins bind quite strongly to the paper
- proteins first denatured in 10% (v/v) trichloroacetic acid
- immersed in solution of a dye that does not stain the
support material
After a PAGE we achieve fractionation of a protein mixture during the electrophoresis process how can we further investigate the fractionations?
how?
with a western blot
- transfer protein from gel to cellulose paper or PVDF via
electroblotting process - probing the blot; antibody, but first incubate blot in a
protein solution - in order to block remaining
hydrophobic binding sites on the cellulose sheet - then incubation in a dilution of an antiserum (primary
antibody) that will bind to antibody - 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 - By careful comparisons of the blot with a stained gel of
the same sample, the protein of interest can be identifi
ed
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
agarose
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: ____
620 000
6.4.1 Agarose Gel Electrophoresis of DNA
What is the reason for seperation in agarose gel?
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.
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?
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.
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
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
