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Cheatography

Electrophoresis Cheat Sheet (DRAFT) by

Electrophoresis Introduction Types

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Electr­oph­oresis

Separation technique
on the basis of charge
incomplete separation
"­mig­ration of a charged particle under the influence of an electric field"
separates- amino acids, peptides, proteins, nucleo­tides, nucleic acids; possess ionizable groups

Points to remember-

Ion
Migrates towards
Cation(+)
Cathode
Anion(-)
Anode

Purpose

To determine number , amount, mobility of components in a given sample OR to separate them
To obtain inform­ation about the electrical double layers surrou­nding the particles

Factors affecting electr­oph­oretic mobility-

Sample
Charge
Higher the charge greater is electr­oph­oretic mobility
Buffer
compos­ition
Type of buffer depend upon what sample is to run
(charge to mass ratio)
 
Charge depends upon pH
   
e.g, formate, citrate, phosphate, EDTA, acetate pyridine, Tris, and barbitone etc
 
Size
Bigger the molecule greater the frictional force
 
ionic strength
between 0.05-0.1M
   
Larger the particle slower the electr­oph­oretic mobility
   
Higher the strength of buffer more the current carried by buffer ions that affect electr­oph­oretic run
 
Shape
Rounded molecules has lesser frictional and electr­ostatic retard­ation compared to sharp molecules
 
pH
Determines degree of ionization of organic compounds
   
Globular and Fibrous proteins
   
Amino acids shows charge based on surrou­nding pH
Electric field
 
rate of migration under unit potential gradient is referred to as mobility of the ion
Medium
led to electr­o-o­smosis
adsorption
   
increase in potential gradient increases the rate of migration
   
molecular sieving
   
current (total charge carried per second to the electrode) in the solution placed between two electrodes is carried mainly by the buffer ions, only a small proportion being carried by the sample ions. An increase in the potent­ial­-di­ffe­rence therefore increases the current.

Advantage over free electr­oph­oresis

Microl­iters of sample are sufficient
Sample components during their migration split up into as many different zones as it contains differ­ently migrating compon­ents. each zone consisting of a single component which can be easily isolated
stabil­izing system does not allow the zones to disperse and spoil the separation
Many detection schemes are Available And elution helps in further analysis

Gel electr­oph­oresis

Separation not only depends on the charge on the molecule but also on its size.
Gels are porous and the size of the pores relative to that of the molecule determines whether the molecule will enter the pore and be retarded or will bypass it.
Resolution of a sample is sharper and better in a gel than in any other type of medium.

Electr­oph­oretic Mobility in Gels

Molecular sieving action of the gels and its effect on the mobility of a macrom­ole­cule.
The pore size thus the molecular sieving action and therefore the effect on electr­Â­op­h­o­retic mobility of a molecule are functions of gel concen­Â­tr­ation
Kr= C (R + r)
Kr is the retard­ation coeffi­cient. C is constant. R is the mean radius of the macrom­olecule and r is the radius of the gel fibers.

Solubi­lizers

To study subunit compos­ition of oligomeric proteins
Solubi­lizers destab­ilize native structure of proteins by destroying charges that associate subunits together
Urea - Disrupt hydrogen bonds At high concen­tration (3-12M). Also, dsDNA can be rendered into ssDNA by use of urea.
SDS - SDS is an anionic detergent and disrupts macrom­ole­cules whose structure has been stabilized by hydrop­hobic associ­ations. Also imparts a large negative charge to the denatured polype­ptides
beta mercap­toe­thanol - Disrupt Disulphide bridges. Separation of peptides in proteins Linked by disulphide bonds

Types of Gel

Starch Gel, Agarose Gel, Acrylamide Gel and Agaros­e-A­cry­lamide Gel

Starch Gel

High porosity starch gels are obtained by using 2% (w/v) starch and low porosity gels are obtained by adding 10-15% starch to the buffer.
The pore size in a starch gel cannot be controlled and this is the biggest drawback of these gels.
Difficult to prevent contam­ination of starch gels by microo­rga­nisms.
Another disadv­antage of starch gels is that upon staining to detect the separated compon­ents, the starch gel turns opaque making direct photoe­lectric determ­ination imposs­ible.
the resolving power of starch gels is very high and can be matched only by polyac­ryl­amide gels. One of their important applic­ations is the analysis of isoenzyme patterns (zymog­rams).
 

Principle

Any charged ion or molecule migrates when placed in an electric field.
The rate of migration depend upon its net charge, size, shape and the applied electric current.

Formula

Electr­oph­oretic mobility (µ)

Electr­oph­oretic mobility (µ) of an ion is used, which is the ratio of the velocity of the ion to field strength (v/E)
Electr­oph­oretic mobility v is directly propor­tional to the charge and inversely propor­tional to the viscosity of the medium, size and shape of the molecule

Formula

Types-

Free electr­oph­oresis

carrie­r-free electr­oph­oresis
matrix­-free electr­oph­oretic separation technique
used to quanti­tat­ively separate samples according to differ­ences in charge or isoele­ctric point.
Two Main Techni­ques- Microe­lec­tro­pho­resis and Moving Boundary Electr­oph­oresis.

Moving boundary electr­oph­oresis

allows the charged species to migrate in a free moving solution in the absence of a supporting medium
Samples are fractioned in a U shaped tube that has been filled with buffer
An electrical field is applied by means of electrodes at the ends of the U tube and Separation takes place as a result of difference in mobilities
Method was very popular for quanti­tative analysis of complex mixtures of macrom­ole­cules, especially protei­ns,­e.g., those in blood plasma.

fid-3

fig-3

Zone electr­oph­oresis

separation technique employing stabil­izing media
electr­oph­oresis in stabilized media

Paper electr­oph­oresis

Filter paper as a stabil­izing medium is very popular for the study of normal and abnormal plasma proteins
Paper of good quality should contain at least 95% of cellulose and should have only a very slight adsorption capacity.
Chroma­tog­raphy paper is suitable for electr­oph­oresis and needs no prepar­ation other than to be cut to size.
Two arrang­ements of paper in paper electr­oph­oresis are horizontal and vertical

Process

Filter paper
Apparatus - Power pack and Electr­oph­oresis cell
Sample applic­ation
Electr­oph­oretic run

fig

fig

Detection and Quanti­tative Assay

Fluore­scence
Ultrav­iolet absorption
Staining
Detection of enzymes in situ

Applic­ations of paper E-

Separating amino acids into acidic and basic
Separation of enzymes in blood
Protein separation in serum
Studying SCA in blood
 

History

Time
Late 18th century
-
-
1931
Scientist
Faraday
Johann Wilhelm Hittorf, Walther Nernst, and Friedrich Kohlrausch
Friedrich Kohlrausch
Arne Tiselius
Experiment
Laws of electr­olysis
To measure the properties and behavior of small ions moving through aqueous solutions under the influence of an electric field
created equations for varying concen­tra­tions of charged particles moving through solution, including sharp moving boundaries of migrating particles
Molecular separation and chemical analysis

Problems

Generation of heat (of the electr­oph­oretic medium) has following effects-
increased rate of diffusion of sample and buffer ions leading to broadening of the separated samples
thermal instab­ility of samples that are rather sensitive to heat.
formation of convection currents, which leads to mixing of separated samples
decrease of buffer viscosity, and hence a reduction in the resistance of the medium

Electr­oen­dos­mosis

electr­oos­motic flow
Cause-
Due to the presence of charged groups on the surface of the support medium.
E.g
Eg: Carboxyl groups in paper, sulphate impurities in agarose, SiOH groups in capillary electr­oph­oresis

Diagram

Points

1. Electr­oos­motic Flow (EOF): Movement of liquid in response to an electric field, toward the cathode.
2. Zeta Potential: Surface charges on the gel form an electrical double layer, creating a zeta potential that drives ion flow.
3. Cation Migration: Cations near the capillary wall migrate toward the cathode, dragging the solvent with them.
4. Electric Field Setup: An anode (positive) and cathode (negative) are placed to create the electric field across the medium.
5. Ion Separation: EOF aids in separating analytes; positive ions move quickly to the cathode, while negative ions are slowed.
6. Applic­ations: Used in capillary electr­oph­oresis for separating molecules like DNA and proteins by charge and size.

Fig-1

Microe­lec­tro­pho­resis

method for determ­ination of zeta potentials
appara­tus­-ca­pillary cell, two chambers that include electr­odes, and a means of observing the motion of particles.
apparatus is filled with very dilute suspension and the chambers are closed.
direct­-cu­rrent voltage is applied between electrodes in the respective chambers.
One uses a microscope to determine the velocity of particles.
Zeta potential values near to zero indicate that the particles in the mixture are likely to stick together when they collide, unless they also are stabilized by non-el­ect­rical factors.
Particles having a negative zeta potential are expected to interact strongly with cationic additives
In modern days this technique is applied only for measuring the zeta potentials of cells such as R.B.Cs, neutro­phils, bacteria etc.

fig-2

Cellulose Acetate Electr­oph­oresis

Advantages
it is chemically pure
cellulose strips are transl­ucent
very low content of glucose.
Cellulose acetate is not very hydrop­hilic and thus holds very little buffer.