Q7: Types of gel
1. Starch
Partially hydrolysed potato starch makes for another non-toxic medium for protein
electrophoresis. The gels are slightly more opaque than acrylamide or agarose. Non-denatured
proteins can be separated according to charge and size. They are visualised using Napthal Black or
Amido Black staining. Typical starch gel concentrations are 5% to 10%
2. Agarose gel
Agarose gels are made from the natural polysaccharide polymers extracted from seaweed. Agarose
gels are easily cast and handled compared to other matrices, because the gel setting is a physical
rather than chemical change. Samples are also easily recovered. After the experiment is finished,
the resulting gel can be stored in a plastic bag in a refrigerator.
Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that
are larger than 200 kDa.[7] Agarose gel electrophoresis can also be used for the separation of
DNA fragments ranging from 50 base pair to several megabases (millions of bases), the largest of
which require specialized apparatus. The distance between DNA bands of different lengths is
influenced by the percent agarose in the gel, with higher percentages requiring longer run times,
sometimes days. Instead high percentage agarose gels should be run with a pulsed field
electrophoresis (PFE), or field inversion electrophoresis.
"Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb
DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in
electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical
polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may
break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1%
gels are common for many applications.
3. Polyacrylamide gel
Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5
to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Pore size is
controlled by modulating the concentrations of acrylamide and bis-acrylamide powder used in
creating a gel. Care must be used when creating this type of gel, as acrylamide is a potent
neurotoxin in its liquid and powdered forms.
Traditional DNA sequencing techniques such as Maxam-Gilbert or Sanger methods used
polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so the
sequence could be read. Most modern DNA separation methods now use agarose gels, except for
particularly small DNA fragments. It is currently most often used in the field of immunology and
protein analysis, often used to separate different proteins or isoforms of the same protein into
separate bands. These can be transferred onto a nitrocellulose or PVDF membrane to be probed
with antibodies and corresponding markers, such as in a western blot.
Typically resolving gels are made in 6%, 8%,
10%, 12% or 15%. Stacking gel (5%) is poured
on top of the resolving gel and a gel comb
(which forms the wells and defines the
lanes where proteins, sample buffer and ladders
will be placed) is inserted. The percentage
chosen depends on the size of the protein that one wishes to identify or probe in the sample. The
smaller the known weight, the higher the percentage that should be used. Changes on the buffer
system of the gel can help to further resolve proteins of very small sizes.
Q8: Define
SDS solution -SDS Solution (10% w/v) is sodium dodecyl sulfate in distilled, deionized water.
SDS is a detergent that is known to denature proteins. It is used in polyacrylamide gel
electrophoresis for the determination of protein molecular weight.
Buffer solution - A buffer solution is an aqueous solution consisting of a mixture of a weak acid
and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid
or base is added to it.
Wells - Indentations at one end of a gel which DNA samples are loaded into. An electric current is
applied to pull DNA samples through the gel to separate them according to their size.
Casting tray - Gel casting apparatus which consists of 3 parts--the tray, the support, and the comb.
The tray is the actual mold which
provides a shape for the gel as it
polymerizes.
