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Sunday, 19 August 2007

SDS PAGE - What each chemical ingredient does?

The importance of Poly Acrylamide Gel electrophoresis in protein research is un questioned. The composition of the gel is complex and the procedure is more an art than science. Precision is the key word in its making. This article provides information regarding the importance of various ingredients of the PAG


Poly acrylamide gel (PAG) had been known as a potential embedding medium for sectioning tissues as early as 1954. Two independent groups Davis and Raymond employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that made it a versatile medium. Poly acrylamide gel separates protein molecules according to both size and charge. It is a synthetic gel, thermo-stable, transparent, strong, relatively chemically inert, can be prepared with a wide range of average pore sizes, can withstand high voltage gradients, feasible to various staining and destaining procedures and can be digested to extract separated fractions or dried for autoradiography and permanent recording. DISC electrophoresis utilizes gels of different pore sizes. The name DISC was derived from the discontinuities in the electrophoretic matrix and coincidentally from the discoid shape of the separated zones of ions (Anbalagan, 1999). There are two layers of gel namely staking gel or spacer gel and resolving gel or separating gel.
Staking gel or spacer gel: It is a large pore poly acrylamide gel (4%). This gel is prepared with Tris buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer. These conditions provide an environment for Kohlrausch reactions, as a result, proteins are concentrated to several fold and a thin starting zone of the order of 19 microns is achieved in a few minutes. This gel is cast over the resolving gel. The height of the staking gel region was always maintained more than double the height and the volume of the sample to be applied.
Resolving gel or Separating Gel: This is a small pore polyacryl amide gel (3 - 30%). The Tris buffer used is of pH 8.8. In this gel, macro molecules separate according to their size. In the present experiment, 8%, 10% and 12% Resolving gel were used for separating different range of proteins. 8% gel for 24 – 205 kD proteins, 10% gel for 14-205 kD proteins and 12% gel for 14-66 kD proteins
The chemical ingredients of the gel are the following
Tris (tris (hydroxy methyl) aminomethane) (C4H11NO3; mw: 121.14). It has been used as a buffer because it is an innocuous substance to most proteins. Its pKa is 8.3 at 20°C and reasonably a very satisfactory buffer in the pH range 7.0 – 9.0.
Glycine (Amino Acetic Acid) (C2H5NO2; mw: 75.07). Glycine has been used as the source of trailing ion or slow ion because its pKa is 9.69 and mobility of glycinate are such that the effective mobility can be set at a value below that of the slowest known proteins of net negative charge in the pH range. The minimum pH of this range is somewhere around 8.0.
Acrylamide (C3H5NO; mw: 71.08). It is a white crystalline powder. While dissolving in water, autopolymerisation of acrylamide takes place. It is a slow spontaneous process by which acrylamide molecules join together by head on tail fashion. But in presence of free radicals generating system, acrylamide monomers are activated into a free-radical state. These activated monomers polymerise quickly and form long chain polymers. This kind of reaction is known as Vinyl addition polymerisation. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires hooking various chains together. Acrylamide is a neuro toxin. It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysis.
Bisacrylamide (N,N’-Methylenebisacrylamide) (C7H10N2O2; mw: 154.17). Bisacrylamide is the most frequently used cross linking agent for poly acryl- amide gels. Chemically it is thought of having two-acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide was preserved at 4°C.
Sodium Dodecyl Sulphate (SDS) (C12H25NaO4S; mw: 288.38). SDS is the most common dissociating agent used to denature native proteins to individual polypeptides. When a protein mixture is heated to 100°C in presence of SDS, the detergent wraps around the polypeptide backbone. It binds to polypeptides in a constant weight ratio of 1.4 g/g of polypeptide. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment becomes a rod like structure possessing a uniform charge density, that is same net negative charge per unit length. Mobilities of these proteins will be a linear function of the logarithms of their molecular weights.
Ammonium per sulphate (APS) (N2H8S2O8; mw: 228.2). APS is an initiator for gel formation. APS was stored at 4°C.
TEMED (N, N, N’, N’-tetramethylethylenediamine) (C6H16N2; mw: 116.21). Chemical polymerisation of acrylamide gel is used for SDS-PAGE. It can be initiated by ammonium per sulphate and the quarternary amine, N, N, N’, N’-tetramethylethylenediamine (TEMED). The rate of polymerisation and the properties of the resulting gel depends on the concentration of APS and TEMED. Increasing the amount of APS and TEMED results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount of initiators shows the reverse effect. It is recommended that lowest catalysts concentrations that will allow polymerisation in the optimal period of time should be used. APS and TEMED are used, approximately in equimoloar concentrations in the range of 1 to 10 mM. TEMED was stored at 4°C.
The following chemicals are used for processing of the gel and the protein samples visualized in it.
Bromo Phenol Blue (BPB) (3’, 3’’, 5’, 5’’-Tetrabromophenolsulph- onephthalein) (C19H10Br4O5S; mw: 669.99).
BPB is the universal marker dye. Proteins and nucleic acids are mostly colourless. When they are subjected to electrophoresis, it is important to stop the run before they run off the gel. BPB is the most commonly employed tracking dye, because it is viable in alkali and neutral pH, it is a small molecule, it is ionisable and it is negatively charged above pH 4.6 and hence moves towards anode. Being a small molecule it moves ahead of most proteins and nucleic acids. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can bind with proteins weakly and give blue colour.
Glycerol (C3H8O3; mw: 92.09). It is a preservative and a weighing agent. Additon of glycerol (20-30 or 50%) is often recommended for the storage of enzymes. Glycerol maintains the protein solution at very low temperature, without freezing. It also helps to weigh down the sample into the wells without being spread while loading.
Coomassie Brilliant Blue (CBB) (C45H44N3NaO7S2; mw: 825.97). CBB is the most popular protein stain. It is an anionic dye, which binds with proteins non-specifically. The structure of CBB is predominantly non-polar. So is usually used (0.025%) in methanolic solution (40%) and Acetic Acid (7%). Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated in the gel can be removed by destaining with the same solution containing no dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic in nature, it is reported to interfere with staining process. Therefore, large volume of staining solution is recommended. Approximately 10 times the volume of the gel.
Butanol (C4H10O; mw: 74.12). Water saturated butanol is used as an overlay solution on the resolving gel.
Beta Mercapto Ethanol (HS-CH2CH2OH; mw: 78.13). BME was procured from LKB, Bromma, Sweden and was stored at 4°C.
References
1. Anbalagan K (1999). An Introduction to Electrophoresis. Ed. Anbalagan K. pub. The electrophoresis institute, Biotech-Yercaud, Salem, India.
2. Sambrook J and Russel DW (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbour, New York.

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