Protein identification by 2D gels uses a combination of isoelectric focusing (IEF) and SDS-PAGE. In IEF, proteins are separated based on their isoelectric point using an acrylamide gel containing an ampholyte pH gradient established by an electric current. Next, SDS-PAGE further separates the proteins by size. Finally, the proteins are visualized after staining, allowing identification by their position on the 2D gel based on isoelectric point and molecular weight.

1. Protein Identification by 2D gels

2.  2D- Gel electrophoresis - combination of two techniques IEF and SDS-PAGE
 Proteins are separated based on charge and size.
 I dimension – carried out in polyacrylamide gels in narrow tubes in the
presence of urea (8 M urea + anionic detergent)
 The denatured proteins are separated according to their isoelectric points.
 The gel will be extruded from the tubes by applying slight pressure to one end
then incubated for 15 min in a buffer containing SDS and placed along the
stacking gel of the SDS added gel.
 The gel is fixed in place by pouring a molten agarose in electrophoresis buffer
over the gel.
 Once the agarose is set,(II dimension) electrophoresis commences and SDS-
bound protein run into the gel, stack and separate according to the size.

3. Isoelectric Focussing
 Separation of molecules according to their different isoelectric point.
 High resolution – Able to separate proteins that differ in their isoelectric points
by as little as 0.01 of a pH unit.
 Horizontal gels on glass plates or plastic sheets.
 Separation is achieved by applying potential difference across a gel that contains
a pH gradient.
 Gradient in the gel is formed by the introduction of ampholytes - complex
mixture of synthetic polyamino-polycarboxylic acids.
 Ampholytes in different pH range
 Wide range – 3-10
 Narrow range – 7-8

4.  pH range is chosen such that the samples being separated will have their pI
values within that range.
 Commercially available ampholytes (Bio-lyte and pharmalyte)
 Thin layer IEF gels (0.15 mm) thick, prepared using a layer of electrical
insulation tape as the spacer between the gel plates.
 IEF is carried out in low % gels to avoid any sieving effect within the gel.
 Ampholyte with a suitable pH range and riboflavin will be mixed with the
acrylamide solution and the mixture is poured over the glass plate containing
spacer.
 Photopolymerization is performed by placing the gel in front of the bright
light.

5.  Photodecomposition of Riboflavin generates a free radical and it initiates the
polymerization. (2-3 h)
 Glass plates will be separated so that the gel will be now placed in one of the
glass plate.
 pH range is chosen such that the samples being separated will have their pI
values within that range.
 Commercially available ampholytes (Bio-lyte and pharmalyte)
 Thin layer IEF gels (0.15 mm) thick, prepared using a layer of electrical
insulation tape as the spacer between the gel plates.
 IEF is carried out in low % gels to avoid any sieving effect within the gel.
 Ampholyte with a suitable pH range and riboflavin will be mixed with the
acrylamide solution and the mixture is poured over the glass plate containing
spacer.

7.  Photo polymerization is performed by placing the gel in front of the bright
light.
 Photodecomposition of Riboflavin generates a free radical and it initiates the
polymerization. (2-3 h)
 Glass plates will be separated so that the gel will be now placed in one of the
glass plate.
 Electrode wicks (3 mm thick) strips of wetted filter paper. (Anode –
phosphoric acid & Cathode – Sodium Hydroxide)
 This will be laid along the length of each side of the gel and a potential
difference is applied.
 Due to this potential difference ampholytes forms the pH gradient between
anode and cathode.
 Power is turned-off and samples will be applied by laying on the gel small
squares of filter papers soaked in the sample.

8.  A voltage is again applied for about 30 min to allow the sample to
electrophoresis off the paper into the gel.
 Later the paper squares will be removed from the gel.
 pH region below the pI will be positively charged and will migrate towards
the cathode and vice versa.
 As they proceed, the surrounding pH will be steadily increasing and therefore
the positive charge on the protein will decrease correspondingly until
eventually the protein arrives at a point where the pH is equal to its isoelectric
point.
 The protein will be in zwitter ion form with no net charge, so further
movement will cease.
 Before staining the gel is first placed into10% trichloroacetic acid, which
allows the proteins to get precipitate and the ampholytes will be washed out.

9.  The gel is stained with CBB and then destained.
 The pI of the particular protein may be determined conveniently by running
a mixture of proteins of known isoelectric point on the same gel.
 Used specifically for separating isoenzymes (the different forms of the same
enzyme often differing by one or two amino acid residues)
 As the proteins are in native form, the enzymes can be detected in the gel by
washing the unfixed and unstained gel by overlaying with agarose
containing substrate.