6 | Electrophoretic Techniques Flashcards
What two types of electrophoresis units are available?
vertical slab and horizontal slab
Why should all electrophoresis be carried out in an appropriate buffer?
To maintain a constant state of ionisation of the molecules being separated. Any variation in pH would alter the overall charge and hence the mobilities (rate of
migration in the applied field) of the molecules being separated
It appears from Ohm’s law that of we increase the voltage we’re getting a greater resistance, i.e. able to accelerate separation – which would result in a corresponding increase in the current flowing. but what problem arises if we increase the current?
Generation of heat given by the Power (P) equation. Telling us that most of the power will dissipate as heat over time, which in of it self is proportional, in addition to current, to distance migrated by ions.
This frictional force that retards the migration of a charged molecule depends on what?
hydrodynamic size
molecular shape
pore size of medium
viscosity of buffer
What does the electrophoretic mobility express?
the ratio of the velocity of the ion to field strength
The current in the solution between the electrodes is conducted mainly by the ___ ions, a small proportion being conducted by the ___ ions
buffer
sample
What effects (4) does heating of the electrophoretic medium render?
- An increased rate of diffusion of sample and buffer ions, leading to broadening of the separated samples.
- The formation of convection currents, which leads to mixing of separated samples.
- Thermal instability of samples that are rather sensitive to heat. This may include denaturation of proteins (and thus the loss of enzyme activity).
- A decrease of buffer viscosity, and hence a reduction in the resistance of the medium.
Explain the phenomenon of electroendosmosis wrt. electrophoretic separation?
due to the presence of charged groups on the surface of the support medium, these groups may ionise generating negatively charges sites where electrolyte cations are attracted to – forming a electrical bilayer
–> thus when an voltage is applied the cations near the cell wall/electrolyte interface will migrate towards to cathode, dragging solvent along
Agarose is a linear polysaccharide of repeted basic units of?
agarbiose (galactose + 3,6-anhydrogalactose)
What type of bond within and between long agarose chains makes the cross-linked structure having good anti convectional properties?
H-bonds.
If we’re to use agarose gel for proteins, which is uncommon because the pore sizes of a 1% agarose gel are large relative to the sizes of proteins. Name one technique that make use of this? with in mind that proteins are required to move unhindered in the gel matrix according to their native charge.
flat-bed isoelectric focussing
The most commonly used buffer for separating DNA, and in the case of RNA, is?
TRIS-acetate containing EDTA ( TAE)
TRIS-borate containing EDTA (TBE)
What crosslinking agent is used to polymerise acrylamide monomers?
‘bis-acrylamide’
In the polymerisation what type of reaction is happening?
free-radical catalysis
How does one initiate the polymerization?
with TEMED
What are horizontal slab gels invariably used for?
isoelectric focussing or immunoelectrophoresis in agarose
What percentage of polyacrylamide are used in SDS-gel electrophoresis?
10-20 % which in addition to rel. large pore size allowing free movement also introduces a sieving effect that contributes to the separation of proteins according to their size, compared to low-percentage gels (e.g 4 %)
Which method is based on the principle of separation of proteins wrt. size?
SDS PAGE
In SDS-PAGE what does glycerol or sucrose do?
provides solution density –allowing the sample to settle
easily through the electrophoresis buffer to the bottom when injected into the loading
well
In SDS-PAGE what 2 compounds are used to allow protein to open up into rod-shaped structure?
beta-mercaptioethanol and DTT
What phenomenon in conventional discontinuous gel electrophoresis is involved when a sample is loaded into a stacking gel – allowing for the protein sample to settle into a sharp band before it enters the main separating gel.?
isotachophoresis
Does negatively charged protein–SDS complexes migrate towards cathode?
no
Does the % of polyacrylamide in the separation gel give different pore sizes?
yes
SDS–gel electrophoresis is often used after each step of a purification protocol
to assess the ____?
to assess the purity
Why is SDS-PAGE not a feasible method if we aim at detecting a particular protein on the basis of its biological activity?
Due to the denaturing condition
In isoelectric focusing gels,
1. Which types of substances is this method ideal to separate?
2.How is separation achieved by
3. How is the pH gradient formed by
4. Following electrophoresis why can’t the gel be directly stained? and with what compound is the gel stained with?
5. IEF is a highly sensitive analytical technique and is particularly useful for studying ______ in a protein, and how does this diffriciate with SDS-PAGE
6. Which types of enzymes is this method particulary useful separating?
- Amphoteric
- applying a potential difference across a gel that contains a pH gradient
- introduction into the gel of compounds known as ampholytes
- because the ampholytes will stain too, giving a totally blue gel. The gel is therefore first washed with fixing solution. This precipitates the proteins in the gel and allows the much smaller ampholytes to be washed out. The gel is stained with Coomassie Brilliant Blue and then destained
- microheterogeneity of a protein, i.e., the protein may exist in different phosphorylated forms (having different charges, that SDS-PAGE can’t separate because no signifi cant effect on the overall relative molecular mass of the protein in these different forms)
- isoenzymes – which are different forms of the same enzyme, often differing by only one or two amino-acid
residues
With 2D electrophoresis, on what 2 principle is the method based on?
IEF: charge (pI)
SDS-PAGE: size (molecular weight)
wrt. dye Coomassie Brilliant Blue R-250 (CBB), what denaturant mixture is used to to precipitate or fix protein in the gel
acid-methanol mixture: 0.1% (w/v) CBB in methanol:water:glacial acetic acid (45:45:10,
by volume) <– acts as a denaturant to precipitate or fi x the protein in the gel, which prevents the protein from being washed out whilst it is being
stained
Although the Coomassie stain is highly sensitive, many workers require greater sensitivity, such as that provided by ______.?
silver staining
In the case of Western blotting, what compound is the usual candidate for blotting?
antiserum (primary antibody)
Transfer of the proteins from the gel to nitrocellulose is achieved by a technique known as _____ in western blotting?
electroblotting
What two component are commonly included, separately in the protein mixture when we first incubate blot?
bovine serum albumin or whey – to block all remaining
hydrophobic binding sites on the nitrocellulose sheet.
After the blot is first incubated in a protein solution, what compound in the dilution is used in the 2nd incubation.
labelled secondary antibody to visualise binding of primary antibody to antigen on the blot.
For the majority of DNA samples, electrophoretic separation is carried out in ____ gels
agarose
All DNA samples should move towards the anode with the same mobility under an applied electric field. However, separation in agarose gels is achieved because of ____ to their movement caused by the gel matrix
resistance
wrt DNA electrophoresis, what’s in the buffer solution?
glycerol
sucrose
Ficoll
What dye that is also included in the sample solvent, can be used in DNA electrophoresis to easier see the sample being loaded and also acts as an marker for the electrophoresis front?
Bromophenol Blue
Why is no stacking gel needed for DNA electrophoresis?
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.
Give one example of an reagent that can be used to stain a DNA electrophoresis gel
SYBRSafe: are molecules that bind between the stacked base-pairs of DNA (i.e. they intercalate)
wrt. DNA sequencing gels, although we have automated methods such as ___ sequencing for routine applications, for some particular applications, such as DNA footprinting, sequencing gels are still used
Sanger dideoxy
DNA sequencing gels is usually made up of____ gel instead of ____, because the final analysis usually involves separating single-stranded DNA molecules shorter than about 1000 nt and differing in size by only 1 nt
polyacrylamide
agarose
The DNA bands of interest can be cut out of the gel and the DNA recovered
by:
(a) electroelution
(b) macerating the gel piece in buffer, centrifuging and collecting the supernatant
(c), if low melting point agarose is used, melting the gel piece and diluting with buffer.
In each case, the DNA is finally recovered by precipitation of the supernatant with ethanol.
In relation to agarose gel methods for DNA that can separate DNA fragments up to 60 kb, what technique is used to up to 2 x 1000 kb?, which essentially, allows the separation of whole chromosomes by electrophoresis
Pulsed-field Gel Electrophoresis
Like that of DNA, electrophoresis of RNA is usually carried out in ____ gels, and
the principle of the separation, based on ___, is the same
agarose
size
Regarding electrophoresis of RNA, in what instance is it feasible to run native RNA?
Following extraction, we need a rapid method for checking the integrity of RNA
Why is it so that one may stain native RNA gel?
Because there will almost certainly be some secondary structure within the RNA molecule owing to intramolecular hydrogen
bonding
However, if the study objective is to determine RNA size by gel electrophoresis, then full denaturation of the RNA is needed, to prevent what?
And why is it so that it’s also necessary to run denaturing gels if the RNA is to be
blotted?
hydrogen bond formation within
or even between polynucleotides that will otherwise affect the electrophoretic mobility
to ensure that the base sequence
is available to the probe
