SDS PAGE
- 2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) is an analytical technique used to separate proteins based on their molecular weight. Electrophoresis is a technique used to separate macromolecules in an electrical field. This technique employs discontinuous polyacrylamide gel for support and SDS for denaturation.
- 3. SDS (also called lauryl sulfate) is an anionic detergent. Its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS masks all the charges present on protein molecules giving the protein a net negative charge.
- 4. SDS molecule 12 carbon tail attached to a sulphate group
- 5. Polyacrylamide is a polymer formed from acrylamide subunits. It is highly water-absorbent, forming a soft gel when hydrated. Polyacrylamide is ideal for protein separations because it is chemically inert and electrically neutral. It forms a mesh like matrix which allows for separation based on weight.
- 6. When proteins are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less resistance from the gel matrix. SDS is a detergent with a strong protein- denaturing effect and binds to the protein backbone in ratio to its mass. In the presence of SDS and a reducing agent(mercaptoethanol) that cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length.
- 7. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because the charge-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
- 9. Gel is polymerized between two glass plates in a gel caster or casting frame, one is short plate and the other is spacer plate. Clamping frame to hold gel cassettes inside the tank. Electrode assembly. Combs for well formation. Power supply.
- 10. The gel used in SDS may or may not be a discontinuous system. Discontinuous system comprises of a 5% stacking and 4-15% resolving gel based on the concentration of TEMED. Typical components of these gels are: o SDS o APS (ammonium per sulphate) o TEMED(tetra methyl ethylene diamine) o Acrylamide bis o Tris (Molarity varies between resolving and stacking gels).
- 11. Hand cast gels can be made in laboratory by the researchers themselves. Align the short plate and stacker plate such that their bottoms are at exactly the same level and clamp them into casting frame. Place casting frame upon casting stand. As a non standard step, water can be poured in between the two plates, to check for a leak which can later be removed by filter paper. Add all the ingredients into a beaker except for TEMED. When the performer is ready to pour the ingredients between the two plates only right before then the TEMED should be added to the rest of ingredients.
- 12. Resolving buffer is poured between the plates first and allowed to stand until solidified. Butanol can be added upon it to produce a straight bubble free layer which can later be removed by filter paper. Once resolving buffer solidifies Remove butanol and start pouring the stacking buffer. Fill until the mixture reaches the top of short plate Ensure absence of air bubbles and insert comb. Allow it to stand until solidified.
- 13. The sample loaded on a SDS gel usually contains: o Laemlli buffer o DI water o Protein sample Laemlli buffer contains: o SDS o Mercaptoethanol o Tris Hcl pH 6.8 o Bromophenol blue o glycerol
- 14. Add all the ingredients to a centrifuge eppendorf. Centrifuge for1 Minute for run down. Heat on thermal cycler or in Hot water at 95 C for 5 minutes.
- 15. Assemble the electrophoresis assembly. Place the gel cassette in the electrode assembly with the short plate facing inside of the electrode assembly. For running a single gel place dummy plate(buffer dam) on the other end of electrode assembly. Remove the comb from gel cassette. Load sample in the well produced by combs using micropipette tips.
- 16. Add running buffer into gel box and fill up to the mark stating two gels or four gels as per requirement. Also add running buffer in between assembly holding two gel cassettes. Place the cap of gel box aligning the color coded electrodes(red to red and black to black). insert the switch into power supply, select the desired current or voltage and press run. Alllow it to run until the sample reaches bottom end of the gel.
- 17. Visualization through Coomassie stain Visualization through Silver staining
- 18. Coomassie stain Silver stain Less sensitive Less complicated Require less steps Stain is poured over gel and left for almost 90 minutes. Later de-staining is done to reveal protein bands. Highly sensitive Complex to perform Requires various steps to produce the final result.
- 19. As the electrophoresis completes gel is removed from the cassette and is placed in fixative solution for 20 minutes. After this fixative is replaced by water which is left for 10 minutes Water is replaced by fresh water which is again left for 10 minutes. Image developing solution then replaces water, which takes 15-20 minutes to visualize protein bands present in sample. Stopping solution(5% acetic acid) then replaces developing solution to stop the reaction.
- 20. Highly sensitive method Very small amount of sample is required. Estimation of protein size. Estimation of protein purity Comparison of polypeptide composition of different samples. Analysis of number and size of polypeptide subunits. Determination of molecular weight.
Editor's Notes
- #2: Need finishing
- #3: SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a variant of polyacrylamide gel electrophoresis, an analytical method in biochemistry for the separation of charged molecules in mixtures by their molecular masses in an electric field.
- #15: Prepare 2X sample buffer consisting of 0.5M Tris-HCl, pH 6.8, 4.4% SDS, 300mM Mercaptoethanol, 10mg/ml Bromophenol Blue and mix with equal volume of sample (For greater reproducibility, employ National Diagnostics preformulated 2X sample buffer, Protein Loading Buffer Blue 2X). Bring to 95° C for 10 minutes, cool to room temperature before loading.
- #19: 50 ml development accelerator 5 ml image development reagent 5 ml reduction moderator 5 ml silver stain complex 35 ml DI h2O
- #20: Fixative soln:Mthanol 60ml, DI h2O 20ml, Acetic acid 20ml ,Image enhancing concentrate 20ml Image developing soln:image developing reagent:5 ml,reduction moderator:5 ml, silver stain complex: 5ml and image development accelerator 50 ml add at the end.