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Poly-acrylamide Gel Electrophoresis
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  • 1. Poly-acrylamide Gel Electrophoresis Mohit Kumar Ram and Jitendra Kumar CoF Mangalore jitenderanduat@gmail.com
  • 2. Gel • Substantially dilute cross-linked system, which exhibits no flow when in the steady-state • Solid, jelly-like material that can have properties ranging from soft and weak to hard and tough • By weight, gels are mostly liquid, yet they behave like solids due to a three-dimensional cross-linked network within the liquid • ExamplesPolyacrylamide gel, Silica gel, Starch gel, Agarose gel etc. jitenderanduat@gmail.com
  • 3. Types of Gels • Organogels • Xerogels • Hydrogels jitenderanduat@gmail.com
  • 4. Organogels • An organogel is a non-crystalline, non-glassy thermoreversible (thermoplastic) solid material composed of a liquid organic phase entrapped in a three-dimensionally cross-linked network • The solubility and particle dimensions of the structurant are important characteristics for the elastic properties and firmness of the organogel • Organogels have potential for use in a number of applications, such as in pharmaceuticals, cosmetics, art conservation, and food • Examplesorganic solvent, mineral oil, or vegetable oil jitenderanduat@gmail.com
  • 5. Xerogels • A xerogel is a solid formed from a gel by drying without shrinkage • Xerogels usually retain high porosity (25%) and enormous surface area (150–900 m2/g), along with very small pore size (1-10 nm) • When solvent removal occurs under hypercritical (supercritical) conditions, the network does not shrink and a highly porous, low-density material known as an Xerogel is produced • ExampleSilica gel jitenderanduat@gmail.com
  • 6. Hydrogels • Hydrogel (also called aquagel) is a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium • Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content(99%) • Hydrogels which are also known as „Smart Gels‟ or „Intelligent Gels‟ • These hydrogels have the ability to sense changes of pH, temperature, or the concentration of metabolite • It is common used gel in laboratories • ExamplesStarch gel, Agarose gel, jitenderanduat@gmail.com gel etc. Polyacrylamide
  • 7. Acrylamide • Acrylamide (or acrylic amide) is a chemical compound with the chemical formula C3H5NO • It is a white odorless crystalline solid, soluble in water, ethanol, ether, and chloroform • Acrylamide is prepared on an industrial scale by the hydrolysis of acrylonitrile by nitrile hydratase • It is carcinogenic as well as Neurotoxic compounds • Most acrylamide is used to synthesize polyacrylamides polymeririsation process • It is used in the manufacture of dyes, Waste water treatment and other monomers jitenderanduat@gmail.com
  • 8. Polyacrylamide • Also called Cross-linked Polyacrylamide • Polyacrylamide is not toxic • Polyacrylamide is a cross-linked polymer of Acrylamide • It is recommended to handle it with caution • It is highly water-absorbent, forming a soft gel when hydrated • Used in- Flocculate or coagulate solids in a liquid - A subdermal filler for aesthetic facial surgery - Polyacrylamide gel electrophoresis - In soft contact lenses etc. jitenderanduat@gmail.com
  • 9. Polyacrylamide gel • It is a white odorless gel, soluble in water • After polymerization of acrylamide it get cross-linked structure • TEMED stabilizes polymerization free radicals and improves • Here, the toxic affect of acrylamide get vanish (95%) • Amount of polyacrylamide salt dissolved (conc.) is directly proportion to cross –linked nature of gel jitenderanduat@gmail.com
  • 10. Polyacrylamide gel Preparation • Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross linking agent N N‟ methylene bis-acrylamide Acrylamide + N N’ methylene bis acrylamide Ammonium persulfate (catalyst) Chemical Polymerization + TEMED (N,N N’ N’ tetramethylethylene diamine) Polyacrylamide jitenderanduat@gmail.com
  • 11. Electrophoresis • Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field • This electrokinetic phenomenon was observed for the first time in 1807 by Reuss • Electrophoretic mobility μe defined as: • Examples- DNA electrophoresis - Gel electrophoresis (SDS-PAGE) - Pulsed field gel electrophoresis( technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying an electric field) etc. jitenderanduat@gmail.com
  • 12. Poly-Acrylamide Gel Electrophoresis(PAGE) • Electrophoresis in which we use polyacrylamide gel as a sieving/filtering material • Poly-Acrylamide Gel Electrophoresis (PAGE) is used for Qualitative Characterization of protein • This procedure is limited to the analysis of protein with a weight range of 14,000-100,000 Da • It is possible to extend the weight range of an electrophoresis gel by various techniques (gradient gel or particular buffer system) jitenderanduat@gmail.com
  • 13. Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis(SDS-PAGE)  It is a type of Poly-Acrylamide Gel Electrophoresis in which, preliminary process is done with help of SDS  The equipment and supplies necessary for conducting SDS-PAGE includes: • An electrophoresis chamber and power supply • Glass plates(a short and a top plate) • Casting frame • Casting stand • Combs jitenderanduat@gmail.com
  • 14. Structure of SDS jitenderanduat@gmail.com
  • 15. Significance of SDS • SDS (sodium dodecyl sulfate) is a anionic detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge • For uniform distribution of charge per unit area(surface)(q/A) • For getting the uniform direction of motion of molecules • If a cell is incubated with SDS, the membranes will be dissolved and the proteins will be soluablized by the detergent jitenderanduat@gmail.com
  • 16. Action of SDS jitenderanduat@gmail.com
  • 17. Procedure of SDS-PAGE jitenderanduat@gmail.com
  • 18. Preparing of Sample • Mix your protein 4:1 with the sample buffer. Heat your sample by either: a) Boiling for 5-10 minutes (Works for most proteins) b) 65ºC for 10 minutes (If you have smearing using the above procedure) c) 7ºC for 30 minutes (Membrane proteins or others that do not enter the gel otherwise may benefit from this type of sample preparation) jitenderanduat@gmail.com
  • 19. Take sample from any part of fish Each sample must contain 50 µg of protein (For example, if you calculated that your protein yield was 5 mg protein /mL (5 µg/µL), you would need 10 µL of that fraction) Place the appropriate volume (based on protein concentration) of each sample into a labeled microfuge tube Add 1/4 volume of 4x Sample Buffer to each sample Place the microfuge tubes containing your sample and sample buffer in a boiling water bath and boil the samples for 2 minutes (Remove the microfuge tubes and place tubes on ice. Chilling the samples keeps them dense so that they “sink” when placed in the wells) jitenderanduat@gmail.com
  • 20. Insert the precast gel to the gel apparatus Add 1x Running Buffer to the buffer chambers of the electrophoresis apparatus Connect the leads to a power supply and electrophorese the samples until the bromophenol blue dye front has traveled to the very bottom of the gel (200 V for ~ 45 minutes) After electrophoresis, carefully remove the fragile gel from between the glass plates, and submerge the gel in Coomassie Blue stain. Shake gently on the shaker for at least 30 minutes Remove the gel from the stain solution and place in Destain I for 15 minutes to 1hr. Remove and put in Destain II 1 - 4 hours until the background is clear Put your destained gel on a piece of saran wrap or in Ziploc bag and photograph it with a digital camera jitenderanduat@gmail.com
  • 21. Chemical Preparation Coomassie Blue Stain • 2.5 g Coomassie Brilliant Blue R, 440 mL methanol, 480 mL water, 80 mL glacial acetic acid, filter before use 5x Electrode (Running) Buffer • 45 g Tris-base (15 g/L) 216 g glycine (72 g/L) 15 g SDS (5 g/L) jitenderanduat@gmail.com
  • 22. 4x Sample Buffer • 4.0 mL distilled water • 1.0 mL of 0.5 M Tris-HCl, pH 6.8 • 0.8 mL glycerol • 1.6 mL of 10% (w/v) SDS • 0.2 m L of 0.05% (w/v) bromophenol blue (Store at room temperature. Immediately before use add 0.4 mL B-mercaptoethanol) Prepare acrylamide gel • Consist of 30% acrylamide, 0.8% bisacrylamide, SDS,and a buffer with an adjusted pH • Store at 4ºC in the dark • The ratio of acrylamide to bisacrylamide can be varied for special purposes jitenderanduat@gmail.com
  • 23. Choose a percentage acrylamide based on the molecular weight range of proteins you wish to separate Gel Percentage(%) Molecular weight Range 7 10 12 15 50-500kDa 20-300kDa 10-200kDa 3-100kDa Destaining I solution (50% methanol, 5% acetic acid, freshly made) Destaining II solution (7% acetic acid, 5% methanol, freshly made) jitenderanduat@gmail.com
  • 24. Staining solution • Dissolve 0.25 g of Coomassie brilliant blue in 45 ml of methanol. Add 45 ml of H2O and 10 ml of acetic acid. Stacking Gel Solution (4% Acrylamide) • • • • • • H2O 0.5 M Tris-HCl, pH 6.8 20% (w/v) SDS Acrylamide/Bis-acrylamide (30%/0.8% w/v) ammonium persulfate (APS) (10% (w/v) • TEMED(Tetramethylethylenediamine) jitenderanduat@gmail.com 3.075 ml 1.25 ml 0.025 ml 0.67 ml 0.025 ml 0.005 ml
  • 25. Data analysis Calculate the Rf (ratio of the fronts) of each protein standard, using the equation: Rf = distance of protein migration/distance of dye front migration. • Using computer, plot the log of the molecular weight on the Y axis and the Rf on the X axis jitenderanduat@gmail.com
  • 26. Importance of SDS-PAGE in Modern Ichthyotaxonomy • To detecting the various diseases of fishes and shellfishes on molecular level • SDS-PAGE denotes technique for identifying genetic variation in fishes at the molecular level • Provides a basis to rearrange the species according to their molecular behavior • Gives very satisfying and accurate result about the protein pattern and their types jitenderanduat@gmail.com
  • 27. REFERENCES • http://www.nature.com/nature physci/journal/v230/n12/abs/physci230092a0.html • http://www.sciencedirect.com/science/article/pii/0003269760900361 • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC343768/pdf/nar004780306.pdf • http://pubs.acs.org/doi/abs/10.1021/ma00178a020 • http://en.wikipedia.org/wiki/Polyacrylamide • homepages.gac.edu/cellab/chpts/chpt4/ex4-1.html • http://www.protocolonline.org/prot/Molecular_Biology/Electrophoresi s/Polyacrylamide_Gel_Electrophoresis__PAGE_/index.html • http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protoco lweek11.html • http://course1.winona.edu/sberg/307/Labs/documents/SDSpage.doc jitenderanduat@gmail.com
  • 28. THANK yoU jitenderanduat@gmail.com