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Study Notes: The Electrophoresis Gel

The electrophoresis matrix/gel forces sample components to separate by size through interstices (pores) and possess the following additional properties:

  • Reduction of convection currents in the buffer
  • Inhibition of sample diffusion allowing the separated components to remain as sharp, discrete bands
  • The gel serves as a solid matrix upon which samples can be fixed and detected at the end of the run.

The polyacrylamide gel
This is formed by the polymerisation of acrylamide in aqueous solution in the presence of small amounts of a bifunctional crosslinker (links at both ends of the molecule) such a methylenebisacrylamide (bis). The polymerisation of these two components together produces a mesh-like crosslinked matrix in 3 dimensions in the gel.

The use of relative amounts of acrylamide and bis leads to gels with different strengths and different degrees of crosslinking. The standard nomenclature to describe this situation is:

T = the total % of acrylamide and bis in the gel
C = the % of crosslinker (bis) in the gel as a percentage of both acrylamide and the crosslinker.

For instance, a 9% (total) acrylamide gel containing a 19:1 acrylamide:bis ratio would have the following values:

T = 9% and C = 5% (that is, 1/20 of the total acrylamide species). This means that 1/20 or 5% of the 9% acrylamide gel is bis (0.45% bis + 8.55% acrylamide = a 9% gel).

This is quite confusing but is often listed in gel recipes and should be understood. Remember that methylenebisacrylamide is also a form of acrylamide.

Just mixing acrylamide and bis will not cause polymerisation. A catalyst is required to initiate polymerisation via free-radical formation. Free oxygen radicals are commonly generated by the use of ammonium persulfate in the presence of TEMED (N,N,N’, N’-tetramethylethylenediamine).

Some interesting aspects of catalytic polymerisation:

  1. Ammonium persulfate must be prepared and used fresh,
  2. TEMED smells like fish,
  3. The presence of excess oxygen will actually inhibit the polymerisation process. De-aeration of the gel solution is often required immediately prior to addition of the ammonium persulfate, and
  4. Acrylamide is a potent neurotoxin and great care must be used in its handling. When polymerised, the polyacrylamide loses its toxicity but still should be handled with caution due to the possible presence on un-polymerised acrylamide in the gel.

Polyacrylamide has certain advantages over agarose (mentioned later). The agarose gel has large pore sizes so molecular sieving (separation by size) will not occur for smaller DNA fragments or most proteins. Further, the ability to alter both the acrylamide and the bis crosslinker in the polyacrylamide gel, allows the pore size to be altered in a very reproducible manner. This results in excellent resolution down to 0.1% of the size of the molecule. For example proteins of Mr as close as 100-kD and 99.9-kD can be resolved using appropriate conditions.

Note the following terms and their definitions:

Mr is a symbol for relative molecular mass, which is the proper term for molecular weight
kD is a measure of Mr and is called the kilodalton. One Dalton = the mass of the hydrogen atom. So a kilodalton is equivalent to 1000 hydrogen atoms.

There are also no batch-to-batch variations in the acrylamide and bis as they are not natural products like agarose.

Changing the T and C values of the gel effects control of the polyacrylamide pore sizes.

  • An increase in T leads to a decrease in pore size in a nearly linear relationship. Higher percentage T gels are used to separate smaller molecules.
  • The relationship of C to pore sizes is more complex.
  • Generally a minimum pore size occurs with C at about 5% (a 19:1 gel).
  • Decreasing C below 5% results in a larger pore size as there are fewer crosslinkers BUT increasing C above 5% also leads to larger pore sizes probably due to bundling of strands in the gel.

Generally researchers have settled on the following C values:

  • Most forms of denaturing DNA and RNA gels, C = 5% (19:1)
  • Most native DNA and RNA gels, C = 3.3% (29:1)
  • For SDS-PAGE of proteins, C = 2.6% (37.5:1).

Note the following terms and their meanings:

Native Form - a molecule in its usual form, eg a coiled and compact protein.
Denatured Form - a molecule that has been changed from its native form, eg an uncoiled and randomly shaped protein.

The following table gives recommended acrylamide/bis ratios, gel percentages and the nucleic acid or protein size ranges suitable for each application.

Polyacrylamide Gel Recipes
Recommended applications are in bold

Acrylamide/bis ratio (C value) Gel % (acrylamide + bis = T value) Native DNA/RNA (bp ie base pairs) Denatured DNA/RNA (bp) Protein (kD)
(kilodaltons)
19:1 4 100-1500 70-500 100-200
19:1 6 60-600 40-400 40-150
19:1 8 40-500 20-200 20-100
19:1 10 30-300 15-150 15-70
19:1 12 20-150 10-100 8-60
29:1 5 200-2000 70-800 >150
29:1 6 80-800 50-500 50-200
29:1 8 60-400 30-300 30-125
29:1 10 50-300 20-200 20-100
29:1 12 40-200 15-125 10-70
29:1 20 <40 <40 <30
37.5:1 6 - - 60-200
37.5:1 8 - - 50-150
37.5:1 10 - - 25-100
37.5:1 12 - - 15-80

Adapted from http://www.nationaldiagnostics.com/electrassistance/electro1_3.html

The agarose gel
Agarose is a natural colloid extracted from seaweed. It is a linear polysaccharide composed of the repeating unit agarobiose. This consists of alternating units of the sugars:

  • 1,3-linked -D-galactopyranose, and
  • 1,4-linked 3,6-anhydro- -L-galactopyranose.

As mentioned earlier, agarose gels have substantially larger pore sizes than polyacrylamide and therefore have poorer resolution but the large pore sizes are of benefit when working with very large molecules such as fragments of DNA - those above about 400 bp. Agarose has the added advantage of being simpler, quicker and safer to prepare than polyacrylamide.

This image shows the molecular structure of agarobiose.
Structure of one Agarose molecule

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