4. Basic Electrophoresis

ELECTROPHORESIS

ELECTROPHORESIS
Electrophoresis is used to separate macromolecules
(Proteins, RNA, DNA, etc) based on their charge.
The NET CHARGE on a molecule will determine
how far it will move in a charged electrical field.
By placing the molecules in wells in the gel and
applying an electric current, the molecules will move

through the matrix at different rates, towards the

anode if negatively charged or towards the cathode

if positively charged.

openwetware.org/.../500px-Be109gelmigration.jpg

ELECTROPHORESIS

Electrical Field

Anode

Cathode

SEPARATION OF PROTEINS

BY

SDS P.A.G.E. GEL ELECTROPHORESIS

SDS P.A.G.E. GEL ELECTROPHORESIS is used to
separate protein molecules.

Proteins
Protein molecules can have either a positive or negative
net charge due to the nature of the amino acid side

chains in their structure.
The net charge is pH dependent and you can end up
with different net charges on the protein at different

pH values.


Some protein amino acid side chains

www.le.ac.uk/.../biochemweb/images/uncharged.gif

Proteins, unlike nucleic acids, can have varying charges
and complex shapes, therefore they may not migrate

into the gel at similar rates, or at all when placed in

an electrical field.
Proteins therefore, are usually denatured in the
presence of a detergent such as sodium dodecyl

sulfate/sodium dodecyl phosphate (SDS/SDP) that

also coats the proteins with a negative charge.
If the proteins were not denatured then differences
in their complex shapes would cause some proteins to

better fit through the gel matrix than others.



Generally, the amount of SDS bound is relative to the

size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio.

Since denatured proteins act like long rods instead of

having a complex tertiary shape, the rate at which the

resulting SDS coated proteins migrate in the gel

is relative only to its size and not its charge or shape.

The denatured proteins are subsequently applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer.

An electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode.

Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty (they encounter more resistance).


Procedure


After a set amount of time (usually a few hours- though this depends on the voltage applied across the gel; higher voltages run faster but tend to produce somewhat poorer resolution), the proteins will have migrated different distances in the gel, based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin.

Thus proteins may be separated roughly according to size (and therefore, molecular weight).


Bromophenol Blue, Coomassie Brilliant Blue or 2-Mercaptoethanol can be

used to visualise the various proteins after the electrophoresis has

finished.

After staining, different proteins will appear as distinct bands within the gel. It is common to run "marker proteins" of known molecular weight in a separate lane in the gel (Lane 1), in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker.

1

2

3

4

5

6

SEPARATION OF RNA and DNA

BY

AGAROSE GEL ELECTROPHORESIS

Electrophoresis is a technique used to separate large molecules (by size) such as RNA and DNA using a specially prepared gel.

The DNA sample is pipetted into slots (wells) at one end of the gel. DNA has a negative charge and is pulled through the gel and towards the positive electrode during electrophoresis.

A gel matrix (agarose) is used as a molecular filter. Smaller DNA molecules pass through the matrix faster than larger ones. DNA is stained with ethidium bromide, which fluoresces under UV light, allowing us to visualise the DNA.

RNA/DNA AGAROSE GEL ELECTROPHORESIS

Gel Electrophoresis

Nucleic acids are negatively charged

phosphate (PO4-) groups

Electrophoresis resolves by size

Agarose is the usual gel matrix

Ethidium bromide/SYBR green stains DNA & RNA

Fluorescent colour under UV illumination

*

RNA/DNA AGAROSE GEL ELECTROPHORESIS

Structure of DNA

cnx.org/.../latest/sugar-phosphate_backbone.jpg

Structure of RNA

www.emc.maricopa.edu/.../farabee/BIOBK/rna.gif

Agarose Gel Preparation

Agarose : fine white powder; polysaccharide (galactose polymer) isolated from seaweed.

1% (w/v) dissolves in Tris-acetate buffer at ~60 C and the solution sets at ~30 C

*

Agarose is dissolved in boiling water and then forms a gel upon cooling. During this process double helixes form which are joined laterally to form relatively thick filaments

Chemical structure of agarose and structure of the polymers during gel formation.

Agarose Gel Preparation

Extraction of DNA/RNA

DNA extraction

Alkaline lysis

Neutralisation

Precipitation of proteins and cell debris

Precipitation or elution using spin column

RNA extraction
Lysis incorporating instantaneous inactivation of RNasesSeparation of contaminating DNAPrecipitation or elution using spin column
*

Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, size.

Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure.

Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.

Extraction of DNA/RNA

DNA electrophoresis apparatus - An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear.

The negative terminal is at the far end (black wire), so DNA migrates towards the camera.

DNA Gel Electrophoresis

Electrophoresis

equipment

Electrophoresis equipment


DNA sample well

Positive electrode

Negative electrode

Agarose gel

Power pack

Conducting buffer solution

Direction of DNA migration

1057

770

612

495

341

Lane M is a size marker:

A commercially prepared solution containing DNA fragments of known sizes, so we can deduce the sizes of our samples. Samples tested are in 1 to 12. Lanes 1 to 5, 9 & 10 contain larger DNA fragments and therefore have not travelled as far through the gel. Lanes 6 to 8 & lane 12 contain smaller fragments that have travelled further. Lane 11 contains no DNA.

A typical gel looks like this:

Direction of DNA migration


M

1

2

3

4

5

6

7

M

8

9

10

11

12

-

+

The gels are stained with fluorescent dyes like Ethidium bromide or SYBR Green, and the bands are visible under UV light.

Their sensitivites ranges are between 100 pg and

1 ng / band.

Because they are intercalating in the helix, the sensitivity is dependent on the size of the DNA fragment and is lower for RNA detection.


Detection of DNA molecules

Determination of DNA size by gel electrophoresis

The relationship between DNA size and mobility is not linear. In order to deduce DNA size, we must use our size marker lanes. We must plot the distance travelled through the gel (mobility) of each size marker against its known size. The size of a DNA molecule is measured in base pairs (bp).

First we measure the distance each size marker has travelled from the sample well (mobility).


M

1

2

3

4

5

6

7

M

8

9

10

11

12

A

B

C

D

E

DNA sample wells

Then we can plot mobility (mm) against fragment size (bp)

Size markerSize (bp)Mobility (mm)A105723B77027.5C61231.5D49534.5E34141.5Chart12327.531.534.541.5DNA size (bp)Mobility (mm)DNA size (bp)1057770612495341Sheet1Mobility (mm)DNA size (bp)23105727.577031.561234.549541.5341Sheet2Sheet3

We can now deduce the size of our unknown fragments. We will deduce the size of the fragments in lane 1 and lane 7. First measure the mobility of the appropriate samples.


M

1

2

3

4

5

6

7

M

8

9

10

11

12

30.5mm

32.5mm

And then, using our plot, for the mobility of each fragment take the

corresponding size.

LaneMobility (mm)Fragment Size (bp)130.5640732.5560
Lane 1

Lane 7

ACKNOWLEDGEMENTS
John N. Abelson (Ed); Melvin I. Simon (Ed); Murray P. Deutscher (Ed)
(Feb 1990). Guide to Protein Purification, Volume 182. Academic Press.

ISBN 978-0122135859.

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4. Basic Electrophoresis