Running of SDS-PAGE Practical
- 1. Practical# 14 29-1-2019 RUNNINGOF SDS-PAGE The separation of macromolecules in an electric field is called electrophoresis. A very common method for separatingproteinsby electrophoresisuses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate(SDS)to denaturetheproteins. Themethod is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its moleculeshave a net negative charge within a wide pH range. A polypeptidechain bindsamountsofSDS in proportion to its relative molecuar mass. The negative charges on SDS destroy most of the complex structure of proteins and are strongly attracted toward an anode (positively-charged electrode) in an electric field. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Becausethecharge- to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the relative abundance of major proteins in a sample, and to determine the distribution of proteins
- 2. among fractions. The purity of protein samples can be assessed and the progress of a fractionation or purification procedurecan be followed. Different staining methodscan be used to detect rare proteinsand to learn somethingabout their biochemical properties. Specialized techniques such as Western blotting, two-dimensional electrophoresis, and peptide mapping can be used to detect extremely scarce gene products, to find similarities among them, and to detect and separate isoenzymes of proteins Materials: To Pour Gels 30% acrylamide 10% SDS 10% Ammonium persulphate(APS)(make fresh each time) TEMED ((N, N, N', N'-tetramethylethylenediamine) 1.5 M Tris, pH 8.8 (resolving gel) 1.0 M Tris, pH 6.8 (stacking gel) 5x SDS Running Buffer (1 L) Tris 15 g Glycine 72 g SDS 5 g Coomassie Blue Stain 10% (v/v) acetic acid 0.006% (w/v) Coomassie Blue dye 90% ddH2O Isopropanol Fixing Solution 10% (v/v) acetic acid 25% (v/v) isopropanol 65% ddH2O
- 3. SDS sample loading buffer (40 ml) ddH2O 16 ml 0.5 M Tris, pH 6.8 5 ml 50% Glycerol 8 ml 10% SDS 8 ml 2-βmercaptoethanol 2 ml (add immediately before use), bromophenol. Procedure: 1.Preparation Of Gels: Resolving Gel: 1. Combine all reagents except the TEMED (N, N, N', N'- tetramethylethylenediamine) for the 15% separating gel. When ready to pourthe gel, quickly add the TEMED 0.004mL TEMED. 2. Mix using a Pasteur pipette, and transfer the separating gel solution between the glass plates in the casting chamber to about 3/4 inch below the short plate. 3. Add a small layer of absoluteethyl alcohol on top of the gel prior to polymerization to straighten the level of the gel and remove unwanted air bubblesthat may be present. Once the gel has polymerized, the ethyl alcohol can be removed by absorption with filter paper. 4. Dry ethanolat RT to pouring the stacking gel. Stacking Gel: The stacking gel concentrates proteins loaded into the sample wells so that they are resolved as a unified "line" once they enter the stacking gel. The reason
- 4. for the lower pH is that this "lower ionic strength implies higher electrical resistance and consequentlya higher electric field, provoking the faster movement of the proteins and of every othercharged particle in the gel 1.Combine all reagents except the TEMED for the 5% stacking gel. 2.Mix using a Pasteur pipette, and transfer the stacking gel solution between the glass plates in the casting chamber. 3.Insert the well forming comb into the opening between the glass plates.Boththe resolving and stacking gels should polymerize within six minutes. 4.Once the stacking gel has polymerized, the comb can be gently removed. The polymerized gel between the short plate and spacer plateforms the "gel cassette". 2.Sample Preparation: ADD Sample along with the sample loadingbuffer as mentioned in the materials.In the buffer SDS is an anionic reagent which gives negative charge to the sample by binding with the amino acids,it also breaks the covalent bonds in proteins. Βmercaptoethanolis a reducing agent it breaks the sulphidebondsin the protein thereby giving proteinsa primary structures
- 5. so that shape and charge donot play role in separation but only the size.Bromophenolis a staining dye it gives blue colorto the sample so that they are visible on the gel.glycerol will add viscosity so that the sample donot float in the buffers and move on the gel. 3.Electrophoresis: Remove the gel cassette from the casting stand and place it in the electrode assembly with the short plate on the inside. Slide the electrode assembly (with the gel cassette) into the clamping frame. Press down on the electrode assembly while clamping the frame to secure the electrode assembly.Pour some 1X electrophoresis buffer into the opening of the casting frame between the gel cassettes. Add enough 1X electrophoresis buffer to fill the wells of thegel. Use a gel loadingtip to pipettesome buffer into each well to ensure cleanliness. When all wells are sufficiently cleaned, slowly pipette 20µL of denatured sample or MW marker into each well. When the gel has been loaded, cover the tank with lid and connect to the power supply at 80V until the dye front reaches the bottom of stacking gel.
- 6. 4.Staining the Gel: Removal the gel from the cassette. The plates are separated and the gel is dropped into a staining dish containingdeionizedwater.After a quickrinse, thewater is poured off and stain added. Staining usually requires incubation overnight, with agitation.0.1% Coomassie Blue dye in 50% methanol, 10% glacial acetic acid. Acidified methanol precipitates the proteins. Staining is usually done overnight with agitation..The dye actually penetrates the entire gel, however it only sticks permanentlyto theproteins. Excessdye is washed out by 'destaining' with Isopropanol fixing Solution. Properly stained/destained gels should display a pattern of blue protein bands against a clear background. The gels can be dried down or photographed for later analysis and documentation. 5.Gel Documentation:
- 7. The gels are observed in the UV OR gel Documentation to see the bands and are compared with the standard or marker to find exact molecular weight of the proteins in the sample.