Week 3 biochem - electrophoretic techniques

Question 1

what is the definition of electrophoresis

Answer 1

it is the movement or migration of charged particles in an electric field.
electrophoresis uses an electrical field to separate charged molecules.

Question 2

what are the biological molecules that have ionisable groups in a solution that has an electric field

Answer 2

the biological molecules are:
- amino acids, peptides and proteins
- nucleotides and nucleic acids

Question 3

what is the nature of the electrical charge dependent on

Answer 3

the nature of the electrical charge is pH-dependant
if you change the pH, the acidity and the basis of the solution, then the electrical charge of the molecule will change.

Question 4

where does the anion and cation migrate to

Answer 4

positively charged molecules which are cations migrate to the negative cathode.
negatively charged molecules which are anions migrate to the positive anode.

Question 5

what equipment is used in electrophoresis

Answer 5

• anode
• cathode
• power pack
• tank, with an aqueous solution, in the solution
• the molecule of interest
• analysts

Question 6

what is the use of the power pack in electrophoresis

Answer 6

it provides electrical power which is used for the movement of charged molecules

Question 7

what is the electrophoresis unit made of, what are the 2 different types of electrophoresis units and why are they used

Answer 7

the electrophoresis unit is made up tank, electrodes, gel, etc.
the 2 different types are:
- vertical (stands-up right) gel system
> separates proteins in acrylamide gels
- horizontal (gel lying down in the tank) gel system
> separates DNA in agarose gel

both vertical and horizontal gel systems operate the same way.

Question 8

what is the point of a buffer solution

Answer 8

it is an aqueous solution that maintains its pH and thus allows the molecule to have a charge and move

Question 9

what causes molecules to have different velocities

Answer 9

molecules have different velocities due to the:
- different electrical charges
- same electrical charge but different molecular sizes and thus different frictional coefficient

Question 10

where are molecules located when electrophoresis is completed

Answer 10

when electrophoresis is completed, molecules that have different velocities will be located at different distances from the electrode.
smallest DNA fragments have the greatest velocities ( as they get through pores quickly)
largest DNA fragments have the least velocity

Question 11

what is the potential difference and what is it measured in

Answer 11

potential difference is the difference in electrical potential energy between the electrodes.
the potential difference allows a current of charged molecules to flow, from the cathode to the anode and anode to the cathode.
it is measured in volts (v)

Question 12

what is frictional coeffiecent

Answer 12

potential difference forces an electrically charged molecule to go towards an electrode but the frictional resistance acts against this force due to the frictional coefficient.
frictional coefficient depends on:
- hydrodynamic size and shape of the molecule (narrow and thin molecules will travel through the gel quickly whereas big bobbed-shaped molecules will have difficulty moving through the gel)
- the pore size of the electrophoresis medium (the gel) (small pores will have greater resistance than large pores
- buffer viscosity (if the buffer is thick and sticky, it will be more resistant than a thin buffer)

Question 13

what is the equation of velocity (of a molecule)

Answer 13

velocity (of a molecule) = (potential difference x charge on molecule)/ (distance between electrode x friction coefficient)

potential difference is the voltage between the anode and cathode
distance between electrodes is how far the molecule has travelled

Question 14

what causes heat when the molecules are going through the gel and why is this a problem

Answer 14

molecules running through the gel generate heat from gel resistance.
the problems that occur when heat is produced are:
- increased molecular diffusion which causes band broadening as things start to move at high speed due to heat
- it creates conventional currents which cause sample mixing due to sample moving lanes.
- heat-sensitive molecules will degrade e.g. protein denaturation
- decreases buffer/gel viscosities which causes fluctuating velocity of molecules which also leads to band broadening

you can decrease the heat in the electrophoresis tank by putting it in a walk-in fridge room.
heating effects can be minimised by controlling the voltage

Question 15

what happens if there are uneven temperatures in the gel

Answer 15

if there are uneven temperatures in the gel then molecules will move faster in the warmer areas in comparison to the colder areas and this causes uneven and slanted bands

Question 16

what is agarose gel and how is it made

Answer 16

it is a linear polysaccharide
how the gel is formed:
- is it dissolved by heating agarose powder in an aqueous solution (concentration range from 1%-3%)
- then the solution is poured into the tray to set
you can control how much agarose to use, 1% agarose will give you big pores whereas 3% agarose will give you small pores.

Question 17

how is the pore size determined in agarose gel

Answer 17

the pore size is determined by agarose concentration
low concentration agarose = large pores
high purity agarose = small pores and you will have low ionic interactions between gel and sample molecule thus high purity agarose lowers the chance of molecule binding to agarose

Question 18

where is agarose gel used and what is its advantage

Answer 18

it is generally used in nucleic acid (DNA and RNA) electrophoresis.
agarose is usually used to separate nucleic acid in electrophoresis.
the advantage of agarose gel is you can cut agarose gel using a scalpel and then you have a blob of RNA or DNA in the gel and you can do anything to it. heating releases sample for analysis.

Question 19

what is polyacrylamide gel and what is it made from

Answer 19

it is usually used to separate proteins
it is a cross-linked polymer made from acrylamide monomers and bis-acylamide, catalysed by sulphate free radicals.

acrylamide monomers can be made to form by reacting with T-med molecules and ammonium sulphate.
this creates a sulphate-free radical which causes the cross-linked reaction

Question 20

how is pore size determined in polyacrylamide gel

Answer 20

pore size is determined by the concentration of acrylamide and bis-acrylamide.
- low concentration (4%)
= large pore size
= DNA separation
- high percentage (10-20%)
= small pore size
= protein separation

Question 21

what is a buffer solution

Answer 21

it is an aqueous solution, it has buffering properties and neutral pH.
it is used to both create a gel and running buffer

Question 22

what buffers are used in different types of separation

Answer 22

DNA separation
TRIS-acetate solution containing EDTA (TAE)

RNA separation
TRIS-borate solution containing EDTA (TBE)
EDTA TBE is better than EDTA TAE because TBE is more stable and provides better buffering capacity for the longer processes of RNA

protein separation
sodium Dodecyl sulfate (SDS)
it is an anionic detergent (a negatively charged detergent)

Question 23

why does the protein denature when using SDS

Answer 23

proteins boiled in SDS, with e.g. B-mercaptoethanol, will denature, this is because, under the stress of being boiled in detergent and B.mercantoethanol l, the proteins will unfold and become 1 long chain

Question 24

what want basis is protein separated when coated with SDS

Answer 24

when proteins are coated with SDS in a negative charge thus protein will only separate on the basis of size and not charge

Question 25

why is loading dye used in agarose gel or polyacrylamide gel

Answer 25

loading dye is used to allow you to see what you are doing when you are loading the sample into the well and it allows you to track the sample as its running through the gel

Question 26

what does the loading dye contain

Answer 26

loading dye contains:
- ionisable tracking dye e.g. bromophenol blue
- density reagent e.g. sucrose, glycerol

Question 27

why is the molecular-weight size marker (ladder) used

Answer 27

when you run gel electrophoresis, a molecular-weight size marker is needed cause it allows you to understand your bands and how big they are and their molecular size.
it gives molecular size/weight estimation of proteins, RNA and DNA.
also, you need the ladder to run in the same condition as your sample to get accurate results.
you need to buy the ladder from a manufacturer

Question 28

what does SDS-PAGE

Answer 28

sodium dodecyl sulfate-poly-acrylamide gel electrophoresis - SDS-PAGE

Question 29

what is SDS-PAGE used for

Answer 29

SDS is used for denaturing and electrical charges to a protein in an acrylamide gel.
it is a common method used for qualitative protein analysis

Question 30

in discontinuous gel electrophoresis, where are samples loaded

Answer 30

samples are loaded into stacking gel wells.
stacking gel has large pores thus all proteins can move freely

Question 31

what kind of bands do you get in discontinuous gel electrophoresis

Answer 31

you get shape, defined gel bands

Question 32

on what basis are proteins separated in discontinuous gel electrophoresis

Answer 32

proteins are separated on the basis of size

Question 33

in single gel electrophoresis, what is the pore size

Answer 33

it has a uniform pore size
pores of all the same size run all the way through the gel

Question 34

why is native (buffer) gel electrophoresis

Answer 34

if you don’t want to denature your protein, you use native gel electrophoresis

Question 35

what gel is used in native (buffer) gel electrophoresis

Answer 35

it uses polyacrylamide gel but the buffer doesn’t contain SDS

Question 36

how is protein separated in native (buffer) gel electrophoresis

Answer 36

the protein is separated according to size and native charge (pH-dependent)

Question 37

how is the position of the enzyme detected in native (buffer) gel electrophoresis

Answer 37

the native (natural) protein structure must be maintained so the enzymes can be detected by biological activity.
the position of the enzyme on the gel is detected by adding the substrate because a coloured product is formed.

Question 38

what is the main advantage of gradient gel electrophoresis

Answer 38

it has a great range of pore sizes in the gel to allow both small and large protein separation instead of having to run 2 different electrophoreses, 1 electrophoresis would be with small pores and 1 electrophoresis with large pores.
how gradient gel electrophoresis is made with different pore sizes is at the top of the gel you have big pores and at the bottom of the gel you have small pores, so as the proteins run through the gel, big proteins get stuck at the top and small proteins go to the bottom thus separating big and small proteins.

Question 39

what is isoelectric focusing gel electrophoresis

Answer 39

it separates proteins according to their individual isoelectric point (PI).
there is a pH gradient on the gel, acidic conditions on the top and basic conditions at the bottom.
every protein has a gel isoelectric point, there PI, it is the point where the pH of the protein is neutral and thus no electric charge and thus the protein stop moving through the gel.

Question 40

how does isoelectric focusing gel electrophoresis work

Answer 40

when you turn on the power pack, under charged conditions the proteins will start moving, positive-charged proteins moved towards the anode and negatively-charged move towards the cathode, when the proteins are moving from the top of the gel to the bottom the pH is changing and eventually they will reach at a point in the gel/ pH where the protein will reach its PI which is where they have no electric charge and thus can’t be moved by the electric field and thus the protein stops moving and this is how the proteins are separated.

Question 41

at what resolution does isoelectric focusing gel electrophoresis separate proteins

Answer 41

it has high-resolution power, it separates proteins with a 0.01 pH units difference in pI value

Question 42

why is isoelectric focusing gel electrophoresis a highly sensitive technique

Answer 42

it is a highly sensitive technique because it detects microheterogeneity in a protein, eg. mono-, di and tri-phosphorylated forms (it has no size difference but different isoelectric points.)
there only needs to be a change in 1 or 2 amino acids in some proteins for a change in the PI value to occur.

Question 43

where is isoelectric focusing gel electrophoresis mostly used

Answer 43

it is mostly used in forensic science
this is because it detects different isoenzymes in biological fluids and the difference in the isoelectric point of the same enzyme everyone has you can identify who’s enzyme you found.

Question 44

what is 2-dimensional polyacrylamide gel electrophoresis

Answer 44

it combines IEF (isoelectric focusing) gel electrophoresis and polyacrylamide gel electrophoresis.

first dimension = IEF-GE (isoelectric focusing) separates proteins according to PI)
the gel contains ampholytes (pH gradient) + 8M urea + non-ionic detergent - which denature and solubilise proteins

second dimension = SDS-PAGE separates proteins according to size

Question 45

how does 2-dimensional polyacrylamide gel electrophoresis work

Answer 45

you set your gel with your pH gradient (which runs from the left to right of the gel), you turn on your power pack, and the proteins will separate and stop at their isoelectric point, after the proteins stop you turn the polyacrylamide gel 90 degrees and you put the electrodes at the top and the bottom of the gel and SDS-PAGE will separate the proteins according to size.
these techniques run consecutively and at 90 degrees to one another.

you further separate with SDS-PAGE because you could have 2-3 proteins with identical isoelectric points but they have different sizes so by doing SDS-PAGE you further separate according to their size.

Question 46

what detection staining is used in 2-dimensional gel electrophoresis

Answer 46

after you run your gel, you need to visualise where things are and thus you need to stain the protein
you need to fix them in place, you can’t have proteins diffuse through the gel because of molecular mixing.
big proteins are fixed in place with methanol:water and acetic acid. this mixture sticks the protein in the position in the gel and then you use CBB (Coomassie Brilliant Blue) and then you get blue bands.
small proteins, you use silver staining and silver (Ag) ions will turn to silver on the protein and then you will get silver and brown bands.

Question 47

what techniques are used to estimate the number of proteins present in 2-dimensional gel electrophoresis

Answer 47

one technique to estimate the number of proteins
scanning densitometry
it measures the transmitted light of protein bands
you can use the density of the bonds

another technique is doing spectrophotometry of proteins cut from gel
you can cut proteins from the gel, dissolve it in a solution and use spectrophotometry to work out how much protein you got in there

Question 48

what type of pores are there in agarose gel electrophoresis

Answer 48

agarose gel electrophoresis is nucleic-acid electrophoresis.
in agarose gel electrophoresis there are big pores because its DNA molecule separation.

Question 49

how is DNA separated in agarose gel electrophoresis

Answer 49

the gel concentration is 0.8% agarose gel to separate DNA
the smallest DNA molecules move the furthest/fastest through the gel

Question 50

how is the gel stained in agarose gel electrophoresis

Answer 50

the gel is stained by SYBR green and viewed under UV light

Question 51

what is pulsed-field electrophoresis

Answer 51

it separates large DNA molecules e.g. whole chromosomes

electric fields are applied alternatively at different angles, therefore DNA molecules align one way and then another. The smaller molecules re-align faster than larger molecules and move through the gel without pausing.

top and bottom you have the anode and cathode and you run the electrophoresis for 5 minutes, then you stop it and change the position running at maybe 45 degrees, what happens is molecules move 1 way and then another, because you progressively change the position of the anode and cathode

Question 52

what is electrophoresis of RNA

Answer 52

it is done to check RNA integrity after extraction
the following process is done to check RNA integrity
it is electrophoresed in 2% agarose gel for 1 hour
if the ribosomal RNA (18s and 28s molecules) are clearly resolved then it means it’s high-quality RNA because the RNA is still intact.
RNA size is determined by electrophoresing denatured RNA on agarose gel

Question 53

what is a capillary electrophoresis

Answer 53

small-scale electrophoresis
Reagents sample, detection - you can separate out very small amounts of drugs
voltages: 10-50 kV
capillary tube: 50-100cm long
run time: 20 minutes

the sample is injected at the anode and the analytes travel towards the cathode at different rates, passing through the UV detector.

applications:
- biomedical analysis
e.g. human disease point mutations in the DNA
- Drug detection in urine or blood serum

Question 54

what is microchip electrophoresis

Answer 54

a microchip is the size of a microscope slide thus microscopic channels are placed in the microchip.
it is ultra-small-scale electrophoresis
it can detect up to 10^-18 of a mole
it has capillary-like channels attached to 2 cm^2 glass plastic or quartz sheets.
voltage: it has volts instead of kilovolts
channels: 50nm wide x 10nm deep
run time: a few seconds
when you start the electrophoresis, you have to keep an eye on it or else the proteins can run off the gel.