Methods of electrophoresis

Question 1

Difference between various methods is

Answer 1

type of support medium
cellulose or cellulose acetate for low molecular weight biochemicals such as amino acids and carbs
polyacrylamide or agarose for larger molecules

Question 2

Polyacrylamide gel electrophoresis positive feature

Answer 2

high resolving power for small and moderately sized proteins and nucleic acids
acceptance of relatively large samples
minimal interactions between migrating molecules and the matric
physical stability of matrix

Question 3

Discontinuous gel electrophoresis characteristics

Answer 3

presence of two gel layers- a lower or resolving gel and an upper or stacking gel
buffers used to prepare the two gel layers are of different ionic strength and each has a different pH
stacking gel has a lower acrylamide concentration so its pores are larger

Question 4

Electrophoresis definition

Answer 4

movement of particles under an electric field

Question 5

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Answer 5

most common means of analyzing proteins
is a detergent which denatures proteins by binding to the hydrophobic regions and essentially coating the linear protein sequence with a set of SDS molecules.
The SDS is negatively charged and thus become the dominant charge of the complex. The number of SDS molecules that bind is simply proportional to the size of the protein therefore charge to mass ratio should not change with size. In solution (water) all diff sized proteins covered with SDS would run at about the same mobility. However proteins are not run in water but in polyacrylamide ( inert polymer). This determines in what molecular weight range the gel will have the highest resolving power. The density and pore size of this polymer ca vary according to how you make it. Thus size of molecule that pass through the matric can vary

Question 6

Agarose gel electrophoresis

Answer 6

simple way of separating large fragments of DNA.RNA from one another by size. It is another type of matrix. DNA/RNA does not need a detergent since it already has a large number of negative phosphate groups which are evenly spaced. Thus with SDS-PAGE the charge to mass ratio is constant.

Question 7

DNA denaturing polyacrylamide gels aka sequencing gels

Answer 7

used to look at smaller DNA molecules with much higher resolutions, the DNA is generally denature via heat and run through a thin polyacrylamide gel that is kept near the denaturing temperature. These gels usually contain additional denaturing compounds such as urea. Two pieces of DNA that differ in size by one base can be distinguished from each other

Question 8

Pulsed field gel electrophoresis

Answer 8

uses electrical field to separate DNA according to size
developed from agarose gel
separating larger DNA molecules
fractionates DNA up to 100Mb , agarose up to 50 kb in size

Question 9

Two dimensional electrophoresis

Answer 9

separate along x and y axis
highly effective means of separating proteins by utilising two important properties- their isoelectric point and their size. The protein mixture is first placed onto a gel and allowed to undergo isoelectric focusing. This separates the proteins based on the pH value at which they have a net charge of zero. This is the horizontal portion of the technique because the proteins move either left or right along the horizontal gel slab. The slab is then placed into an SDS-PAGE apparatus which begins to separate the proteins based on size. Since the movement is down along the y-axis we call this the vertical component of the setup. Movement in the SDS-PAGE is perpendicular with respect to the movement of the proteins in the isoelectric focusing setup.

Question 10

Capillary electrophoresis

Answer 10

separates ions based on their electrophoretic mobility dependent on charge of molecule, viscosity and atom radius. Separates based on size to charge ratio in the interior of a small capillary filled with an electrolyte.
Ions of same charge, smaller particle has less friction and faster migration
ions of same size, the one with greater charge moves faster

Question 11

Immunoelectrophoresis

Answer 11

separation and identification of proteins based on difference in electrical charge and reactivity with antibodies

Question 12

Gel electrophoresis applications

Answer 12

forensics
molecular biology
genetics
microbiology
biochemistry

Question 13

Southern blotting

Answer 13

transfer of DNA restriction fragments from agarose gel to cellulose nitrate (nitrocellulose) membrane prior to the hybridization of the resulting blot-immobilised DNA to radioactively labelled probes

Question 14

Process of Southern blotting

Answer 14

prepare DNA mixture by breaking it into small fragments using a protein called restriction enzyme. The mixture of DNA fragments is separated according to size by gel electrophoresis. The double stranded pieces of DNA are denatured or separated into single strands within the gel. DNA is transferred from the gel onto blotting membrane. Membrane carries all bands originally on gel. Membrane is treated with a small piece of DNA or RNA called a probe which has been designed to have a sequence complementary to a particular DNA sequence in sample this allows the probe to hybridize or bind to a specific DNA fragment on the membrane. The probe has a label which is radioactive atom or fluorescent dye. Probe permits detection

Question 15

Northen blotting

Answer 15

same as southern except its RNA we want to separate

Question 16

Western blotting aka protein immunoblot

Answer 16

detect specific protein molecules from a mixture of proteins
can also evaluate size of protein of interest and measure amount of protein expression
often used as follow up test to confirm the presence of an antibody and help diagnose a condition e,g in HIV and Lyme disease testing

Question 17

Process of Western blotting

Answer 17

prepare protein sample by mixing it with SDS detergent which makes proteins unfold into linear chains and coats them with negative charge
protein molecules are separated according to size using gel electrophoresis. The proteins are transferred from gel onto a blotting membrane. Once transfer is complete the membrane carries all the protein bands originally on the gel. Membrane goes through a treatment called blocking which prevents any nonspecific reactions from occurring. The membrane is then incubated with an antibody called the primary antibody which specifically binds to the protein of interest. An unbound primary antibody is washed away and the membrane is incubated again with a secondary antibody that specifically recognizes and binds to primary antibody. The secondary antibody is linked to a receptor enzyme that produces color or light which allows it to be easily detected and images