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ELECTROPHORESIS
ELECTROPHORESIS
Electrophoresis is used to separate macromolecules(Proteins, RNA, DNA, etc) based on their charge.
The NET CHARGE on a molecule will determinehow far it will move in a charged electrical field.
By placing the molecules in wells in the gel andapplying 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
SDS P.A.G.E. GEL ELECTROPHORESIS is used toseparate protein molecules.
Proteins
Protein molecules can have either a positive or negativenet charge due to the nature of the amino acid side
chains in their structure.
The net charge is pH dependent and you can end upwith different net charges on the protein at different
pH values.
SDS P.A.G.E. GEL ELECTROPHORESIS
Some protein amino acid side chains
www.le.ac.uk/.../biochemweb/images/uncharged.gif
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 thepresence 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 differencesin their complex shapes would cause some proteins to
better fit through the gel matrix than others.
SDS P.A.G.E. GEL ELECTROPHORESIS
SDS P.A.G.E. GEL ELECTROPHORESIS
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).
SDS P.A.G.E. GEL ELECTROPHORESIS
Procedure
SDS P.A.G.E. GEL ELECTROPHORESIS
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).
SDS P.A.G.E. GEL ELECTROPHORESIS
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 Gel Electrophoresis
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
DNA Gel Electrophoresis
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.
DNA Gel Electrophoresis
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).
DNA Gel Electrophoresis
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)
DNA Gel Electrophoresis
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)23105727.577031.561234.549541.5341Sheet2Sheet3We 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.
DNA Gel Electrophoresis
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.
DNA Gel Electrophoresis
LaneMobility (mm)Fragment Size (bp)130.5640732.5560Lane 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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Mobility (mm)
DNA size (bp)
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Mobility (mm)
DNA size (bp)