Electrophoresis

What is electrophoresis

Movement of charged particles by an external electrical field

The rate of migration of an analyte during electrophoresis is dependent on what properties

Support media properties, electric field strength, temperature

Use of electrophoresis in the clinical lab

DNA and Protein analysis

Common support media of gel electrophoresis

Agarose

How do samples move through agarose for electrophoresis

Molecules migrate through the pores of the agarose (pore size determined by agarose concentration)

How to proteins separate in serum protein electrophoresis

Separate based on mass to charge ratio

Benefits of agarose as support media

Easy to handle, no charge, minimal contribution to electroendosmosis, low protein affinity, clear when dried, allowing for densitometry

What causes electroendosmosis

Support media is often negatively charged, with the buffer often having positive ions. When voltage applied, positive ions move toward cathode (-), bringing water (and solutes in water) with it.

Effect of electroendosmosis

Effect of the net movement of buffer can slow down or reverse direction of analyte migration

Effect of buffer pH

Determines net charge of analytes during electrophoresis

Effect of too low voltage on electrophoresus

Decreased resolution, broad bands, macromolecules diffuse into gel

Effect of too high of current on electrophoresis

Causes excessive heat --> protein denaturation, and produces convection currents in the buffer, causing warped electrophoresis patterns

Effect of ionic strength on electrophoresis

Alters voltage (in constant current system)

- increased = reduced migration rate, but also increases heat

Effect of voltage on electrophoresis

Migration rate is proportional to voltage

Effect of temperature on electrophoresis

High temp = denatured proteins

Low temp = decreased migration rate

Effect of time on electrophoresis

Resolution of bands increases with time

Effect of support media on electrophoresis

Electroendosmosis and pore size effect migration ratw

Why do DNA and protein move through electrophoresis based on mass to charge ratio, and now size?

Pore size isn't small enough to allow for sieving

Mass to charge ratio of DNA

1:1. Only one negative charge (phosphate) on DNA molecule

At a proteins isoelectric point, what is it's charge?

No net charge

Why is plasma protein electrophoresis carried out at alkaline pH

pH > pi = protein has a negative charge, and will travel to the anode

Fastest to slowest electrophoresis movement of plasma protein

Albumin, alpha 1, alpha 2, beta 1, beta 2, gamma

Commonly used stains for SPE

Coomassie Brilliant blue, Amido Black, Ponceau S.

How are SPE zones quantitated

Densitometry

How to visualize nucleic acids in agarose gels

Fluorescent dye, and photographed with UV light source

Cause of unequal SPE migration across well

Dirty electrodes, uneven wetting of gel

Cause of distorted SPE protein zones

Bent applicator, bubble introduced during sample application, too much sample applied

Cause of unusual SPE bands

Hemolyzed = increased alpha 2 and beta

Plasma sample = increased beta 2 and gamma

Medication = unusual albumin migration

Benefit of capillary electrophoresis over gel

Decreased turn around time

Electrophoresis capillary features

Silica tubes, reinforced with polyimide exterior coating/ Gap at the cathodic end to allow for analyte detection

Why can capillary electrophoresis occur at high voltage (hence, run fast)?

Narrow glass tubes of capillary efficiently dissipate heat

Electrokinetic injection

High voltage applied to sample, and kinetic energy drives sample into capillary

Hydrodynamic injection

End of capillary placed in sample, and differential pressure is applied

Which way to proteins flow in capillary SPE?

Negative proteins flow towards cathode (negative) due to strong electroendosmosis force. Injected into capillary from anode

How is DNA injected into capillary electrophoresis

Electrokinetic injection

Polymer in capillary DNA electrophoresis

polyacrylamide at an alkaline pH

Which way does DNA flow in DNA capillary electrophoresis

Injected at cathode (-), and moves toward anode (no electroendosmosis)

How does DNA separate in capillary DNA electrophoresis

As all nucleic acids have same mass to charge ratio, polymer acts as sieve to seperate nucleic acid based on length

How are nucleic acids read in capillary DNA electrophoresis

DNA is labelled prior to sepration, at anode they pass by fluorescent light source and a detector

Common buffers of DNA gel electrophoresis

Tris-acetate-EDTA (TAE)

Tris-borate-EDTA (TBE)

Components of DNA gel electrophoresis loading buffers

Dyes (bromothymol blue and xylene cyanol), and either ficoll, glycerol, or sucrose

Use of dyes in DNA gel electrophoresis loading buffer

Add color to DNA, aiding loading. Also migrates to anode at predictable rate and can be monitored to ensure DNA sample has migrated sufficiently

Use of Ficoll, glycerol, or sucrose of DNA gel electrophoresis loading gel

Increases density of DNA to ensure DNA sample sinks in gel well and is not lost in the buffer

What dye is used to visualize nucleic acids in gel electrophoresis, and how does it wor?

Ethidium bromide - intercalating dye, binding between complementary base pairs of dsDNA. The fluorochrome is excited by UV light and is observed as red light

Issues with ethidium bromide

Mutagen, toxin, suspected carcinogen and teratogen

What is a DNA ladder

Molecular weight markers/standards which are composed of nucleic acid fragments of known sizes and concentrations

Use of DNA markers

- Calculate sample MW

- monitor process of electrophoretic run

- Estimate sample concentration

Before DNA is run on capillary gel electrophoresis, what must be done?

Sample must be labelled with fluorescent tag via PCR, and PCR must be cleaned to remove labels and reagnets