Uploaded byNS-Graphics
SDS PAGE (Electrophoresis) Book Notes
The document elaborates on the process of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a technique used to separate proteins based on their molecular weight. It discusses components necessary for gel preparation, the functionality of SDS in protein denaturation, and the setup of electrophoresis apparatus. Additionally, it outlines the advantages and disadvantages of polyacrylamide gels and provides protocols for gel assembly, sample preparation, and protein staining.
EducationâŠ
More Related Content
Similar to SDS PAGE (Electrophoresis) Book Notes
SDS PAGE (Electrophoresis) Book Notes
- 1.
- 2. Elecirophoresis initiator, ammonium per sulphate(APS), (Chemical method). These components induce p lvmerization. Modification can be made according to the requirement of slong with acrylamide and appropriate buffer generate the free radicals needed to Dplication bychangingthe amount of acrylamide (C,H;No). For larger pore size, size, the ooncentration of monomer and/or cross linker can be increased e.g. to the amountof acryamnide and/or cross linker can be decreased andfor smaller pore esolve (separate)small protein of 8,000 Dato 6000 Da, small-pore-size gel required r15-20%acrylamide) and for larger protein (15,000 Da-13000 Da), larger pore size gouldbe required (7.5-10% acrylamide). Thus poly acryamide gel electrophores is RCCOrdingtotheir size. Three dimensional mesh of poly acryamide gel helps to 'catch' Horksin a similar fashion as an agrose gel, by separating protein molecules the protein molecules asthey are transported bythe electric current. The negatively charged protein molecules are pulled to the positive end but they encounter by the resistancefrom this polyacrylamide mesh. The smaller molecules migrate faster than thelarger molecules (fig. 6). Negative Electrode Stacking. gel Running gel 2 Positive Electrode Lower buffer reservoir Load samples here Buffer big proteins small proteins Advantage of Polyacryamide gel Fig. 6. Electrophoresis : Migration of protein molecules in SDS-PAGE, Well Although the poly acryamide gels are more difficult to prepare and to handle, It ischemically stable and a cross linked gel Glass Plates involving longer time for preparation than agarose gels but they have greater resolving power and can accomnodate larger quantities of sample. We get sharp bands ofseparated molecules Spacer lt is good for separation of lowmolecular weight molecules 243
- 3. 244 Disadvantages 1. Acryamide isatoxic manomer 2. Gels are tedious to prepareand oftenleak 3. We need new gel for new experiment Instrumentation : 1 SDS-PAGE generallyconductsin a vertical gel elctrophoresis(apparatus). We need the following main components(fig. 7). Ithastwobuffer chambers, upper and lower chamber. Bothchambersarefitted with platinum electrode connected to external powersĂșpply(DCvoltage) Gel cassette Loading wells for sample application Clamp to secure gel Upper electrode Gel slab ( Direction of Electrophoresis (+) Lower electrode Tank Supports for bottom of get Modern Phhytotechniques and Biostatistics hrrn Comb Wellforming termplale Glass plates Spacers Electrode Upper buffer reservoir Lover buffer reservoir Fig. 7. Electrophoresis : Different components of PAGE Power supply (DC)
- 4. Iohoresis 2. 4. 5 Gel cassettes spacer, gelcaster etc. Tank AGE. ettes(platesboth notched and baseplate)comb, Power supply Micropipettes,clamps, gloves, tips SDS-PAGE 6. Measuring cylinder, flasks, distilled water, water bath etc. as mentioned earlier performs as 245 (onvented by Laemmli) to separate proteins. Inthis system1/3of the length of gel is alled stacking gel which includes (table 2) the sample loading wells and consists 4%acrylamide preparedin Tris HCl buffer of about pH 6.8 (larger pore size). The remaining2/3 part of the gel length is called'separating or resolving gel and it consistsof acryamide in a concentration of 7.5-20% (depending on the molecular SIzes of the proteins to be separated) prepared in a Tris HCI buffer of pH 8.8 (table3) for smaller pore size. Advantage of this system is that the resolution is improved when proteins run through the stacking gel giving sharper bands than discontinuous system 1. Acrylamide stock solution (30%): Dissolve 29.2 gm of acrlyamide and 0.Ă° gm of N,N"-methylene-bisacrylamide in 100 ml of distilled water. Flter it with 0.45 um. pore size membrane. Acrvamide concentration determines the pore size (table 4) 2. To prepare 1.5 MTris-chloride (pH 8.8) for separating (resolving) gel : Dissolve 18.15 g of Tris (tris hydroxymethyl amino methane) in 50 ml of distilled water, adjust pH to 8.8 with HCl, raise yolume to 100 ml with distilled water. 8. Toprepare 1.0 MTris-chloride (pH 6.8): Dissolve 12.08 gm ofTris in 60 ml distilled water, adjust pH to 6.8 with HCl and raise to 100 ml. 4. 10% SDS: Dissolve 1 gm SDS in 5ml of distilled water and raise the final volume to 10 ml. 5. Gel running buffer :Dissolve 14.4 glycine, lg. SDS in 1lt.of distilled water, adjust pHto 8.3ĂŸy addingsolidTrisand make thefinal volume equal to1lt. 6. Prepare Ammonium Sulphate (APS) 10% in the last and add to induce polymerization process : Dissolve 500 mg of solid APS in 5 ml of distilled water. I. For staining protein Coomassie Brilliant Blue R 250 :Dissolve CBBR-250 (600 mg) in 80ml. methanol, add 20 mlofglacial acetic acid and make it 200 ml with distilled water. Silver staining can also be used.
- 5. 246 8. 9 Destining solution :Mix 400 ml of methanol, 100 ml glacial acetic acid and 500 ml distilled water. Protein sample 10. Marker proteins (Molecules of known molecular weight to estimate the molecular weight of separated proteins inbands). Acrylamide in Resolving Gel |Component (ml) Table 2: Formulationfor SDS-PAGE separating and stacking gels Distilled water TEMED 30% acrylamide solution 1.5 M Tris chloride (pH 8.8) 10% (wt/vol) SDS 10% (wt/vol) ammonium persulphate 7.5% |Component Distilled water 9.6 5.0 0.008 TEMED 5.0 0.2 0.2 |30%acrylamide 1.0M Tris chloride (pH 6.8) 10% (wt/vol) SDS 5.0 7.5 10% %ofacryamide 7.9 6.7 5.0 10.0 0.2 0.008 10% ammonium persulphate 20.0 0.2 Modern Phytotechniques and Biostatistcs 12% 6.6 8.0 5.0 Table 3: 4% Acrylamide Stacking Gel for SDS-PAGE 0.2 0.008 0.2 repare the amnmonium per sulnhatefresh and add in the last to induce the POyinenzation process (polymerization of the resolving gel willtake aPproximatel 30 min). Volume (ml) 7.5% get standard for proteins of 10-300 Kda. 3.6 3.0 2.7 2.6 0.67 1.8 0.5 0.04 0.004 Pore diameter (nm) 0.04 Table 4:Percentage of acrylamidewith respective pore size 15% 4.6 10.0 Method : SDS-PAGE kitare commercially available 5.0 0.2 0.008 0.2 20% 2.7 11.9 5.0 0.2 0.008 0.2
- 6. gactrophoresis 1. 2 3. 4. 5. Assemble the gel electrophoresiskitas per instructions given in the manual supplied with kit and sealthe bottomof plates. A Preparethe resolving (running) gel solutions(as per recipegivenin table 2.) Pourfreshly mixed resolving gel solution into spaces between parallel glass gel-forming plates,leaving at least 1/3space at thetopforthe stacking gel(to be added later). Allowthe gel to polymerize for about 30 minutes at room temperature. A line will becomne visible at thetop for the gel asit polymerizes. Over laythe gel with 1-2 mm of distilled water saturated n-butanoltoexclude O, andensure afresh interface between both gels. 247 Prepare the stacking gel(as per recipe given in table no. 3). Mix the solution gently and pour this stacking gel intothe space abovethe resolving gel. Insert the comb up to 5 mm to create samnle wells in the stacking 80 immediately and allowthe gel to polymerize for at least 30 min. Now remove the bottom spacer from the plates and place the gel in the plates. 1 Add running buffer (glycine buffer) to the chambers at the bottom and top of the gel. Gently remove the comb from between the plates. Avoid bubble formation. The well in the stacking gel portion shouldbe uniformlyfilled with running buffer. There should be no leakage between two buffer chambers. 8. Prepare the standard and unknown protein sample with known volume of buffer in micro centrifuge tube, mix it gentlv and heat at 100°C for 5min in a water bath to make the protein denatured. Now allow the sample tocool at room temperature. 9. Load the sample on to the gel with the help of a syringe carefully. 10. Connectthe electrodes ofthe apparatus andapplyaconstant200-VDC. Until the bromophenol tracking dye reaches near the bottom. 11. Turn off the power supply, remove the gel from the apparatus and separate theplates. 12. Once the SDS-PAGE gel is run, a fixative acid 25% in water or 20% trichloro aceticacid) isadded tofix the proteins so that they do not come out ofthe gel during staining because it keeps proteins as denatured. 13. Carefully transfer the resolving gel to atray filled with 50 ml Coomassie Brilliant Blue (or silver stain) staining solution and let it soak in the solution for 1 hour. 14. Discard the staining solution and place the gel in destaining solution for one hour and dry. Different proteins willappear as dark blue bands on the transparent gel after detaining according to their sizes (and therefore by molecular weight, fig. 8).
- 7. 248 Glycine buffer, pH 8.3 Sampleof mixed proteins, pH 8.3 Stacking gel. pH 6.8 Resolving gel, pH 8.8 Glycine buffer; pH 8.3 Analysis and Detection (a) 1. 2. 3. 4. (b) 1. For each protein band measure the distance from the bottom of the well to the centre of the band. 5. 2. Measure the distance from the bottom of the well to the centre of dye front. 6. Proteins beginning tostack Rf of band = SDS-PAGE has the following applications Fig. 8 Electrophoresis :Migration of proteins through stacking and resolving gels during SDS-PAGE. Calculate the Rf value for each protein band as The molecular weight of a protein in the band can be estimated by comnparing it with the marker proteins of known molecular weight. The separated proteins can be cut from the gel and further analyzed by other proteomic techniques. (c) distance travelled by the protein band 1 distance travelled bythedye front Identification of proteins Modern Phytotechniques and Biostatistics To determine peptidelprotein MW. Bands of protein Determination of sample purity (d) Blotting applications (Southern Blotting) Quantification of proteins Identify existence of disulphide bonds. Resolving proteins