Identification of proteins by 2D gel

Gel Electrophoresis

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.

6. 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.

7. 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.

8. 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.