1. Page 1 Polyacrylamide Gel Electrophoresis(PAGE) Mr. R. K. Lodha

2. Why Use Polyacrylamide Gels to Separate Proteins? Polyacrylamide gel has a tight matrix Ideal for protein separation Smaller pore size than agarose Proteins much smaller than DNA –Average amino acid = 110 daltons –Average nucleotide pair = 649 daltons –1 kilobase of DNA = 650 kD –1 kilobase of DNA encodes 333 amino acids = 36 kD

3. Why polyacrylamide used for a gel?  Chemically inert  Electrically neutral  Hydrophillic  Transparent for optical detection

4. Acrylamide Page 4 Acrylamide CF- C 3 H 5 NO White odourless crystalline solid, soluble in water, ethanol,ether & chloroform Preparedonindustrialscalebythehydrolysis of acrylonitrile by nitrile hydratase carcinogenic as well as Neurotoxic compound. usedinthemanufactureof dyes,Wastewater treatment and other monomers

5. Polyacrylamide Page 5 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.

6. Polyacrylamide gel Page 6 It is a white odorless gel, soluble in water After polymerization of acrylamide it get cross-linked structure TEMED stabilizesfreeradicalsandimproves polymerization Here, the toxic affect of acrylamide get vanish (95%) Amountofpolyacrylamidesaltdissolved(conc.)is directly proportion to cross –linked nature of gel

7. ▶ Polyacrylamide gels are characterized by two parameters: 1. total monomer concentration (%T, in g/100 ml) 2. weight percentage of cross linker (%C). ▶ Higher %T - smaller pores. ▶ The practical ranges for monomer concentration are stock solutions of 30-40%

8. Gel Types Page 8 Agarose Polysaccharide extracted from sea weed. Gel casted horizontally Non-toxic. Separate large molecules Commonly used for DNA separations. Staining can be done before or pouring the gel. Polyacrylamide Gel Cross-linked polymer of acrylamide. Gel casted vertically. Potent neuro-toxic. Separate small molecules. Used for DNA or protein separations. Staining can be done after pouring the gel.

9. A. On The Basis of Supporting Media 1.Slab Gel A.Horizontal B.Vertical 2. Tube Gel B. On The Basis of Types of Separation 1.No-Denaturing/native (Separation by size and charge; charge/mass and shape) 2.Denaturing/Non-Native (separation by size) 3.Other Types (IEF, 2-D-GEL) TYPES OF GEL ELECTROPHORESIS

10. PAGE Page 10

11. Poly Acrylamide Gel Electrophoresis Page 11 It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support matrix. Gels are made by free radical-induced polymerization of acrylamide and N,N’- Methylenebisacrylamide. It is the most widely used technique of electrophoresis.

12. TypesOf PAGE Page 12

13. No denaturing agents Proteins separated based on size, charge and shape. Used when want to keep protein active to study conformation, self-association or aggregation, and the binding of other proteins Native PAGE Page 13

14. Principle  Electrophoretic migration occurs because most proteins carry a net negative charge in alkaline running buffers.  The higher the negative charge density (more charges per molecule mass), the faster a protein will migrate.  At the same time, the frictional force of the gel matrix creates a sieving effect, retarding the movement of proteins according to their size and three-dimensional shape.  Small proteins face only a small frictional force while large proteins face a larger frictional force.

15.  Thus native PAGE separates proteins based upon both their charge and mass.  Because no denaturants are used in native PAGE, subunit interactions within a multimeric protein are generally retained and information can be gained about the quaternary structure.  In addition, some proteins retain their enzymatic activity (function) following separation by native PAGE. Thus, it may be used for preparation of purified, active proteins.

16. Type of Native PAGE Native or Non-Denaturing 1.Continuous System: Both gels and electrophoresis tank have same buffer composition (Single phase gel; a resolving gel)….e.g DNA Agarose gel electrophoresis 2.Discontinuous System: Both tank and gels have different buffers (two phase gel; a stacking gel and separating or resolving gel) e.g. Polyacryl amide gel electrophoresis (PAGE) for proteins

17. SDS- PAGE Page 17 It is a modified version of PAGE whereby Sodium-dodecyl-sulphate (SDS) is used. SDS is an amphipathic surfactant. It denatures proteins by binding to the protein chain with its hydrocarbon ‘tail’, exposing normally buried regions and ‘coating’ the protein chain with surfactant molecules. The polar ‘head’ group of SDS adds an additional benefit to the use of this denaturant.

18. Structure ofSDS Page 18 (NaC12H25SO4) also called sodium lauril sulfate or sodium lauryl sulfate

20.  Mercaptoethanol will break the disulphide bridges.  SDS binds strongly to and denatures the protein.  Each protein is fully denatured and open into rod- shape with series of negatively charged SDS molecule on polypeptide chain. SDS is an anionic detergent. The sample is first boiled for 5min in buffer containing 20 Beta- Mercaptoethanol SDS

21.  On average, One SDS molecule bind for every two amino acid residue.  Hence original native charge is completely swamped by the negative charge of SDS molecule.  Also referred as Discontinuous gel electrophoresis. 21

22. SDS-PAGE Page 22 WHY ? ? ?

23. In their native form, proteins fold into a variety of shapes, some compact, some elongated. The rate of migration of native proteins through a sieving medium is therefore more a reflection of their relative compactness, and less an accurate measure of molecular weight. Denaturing the proteins nullifies structural effects on mobility, allowing separation on a true charge/mass ratio basis. It also separates subunits in multimeric proteins, allowing analysis of large, complex aggregates.

24. BEFORESDS AFTER SDS

25. Principle In denaturing PAGE protein samples are heated with SDS before electrophoresis so that the charge-density of all proteins is made roughly equal. Heating in SDS, an anionic detergent, denatures proteins in the sample and binds tightly to the uncoiled molecule. Usually, a reducing agent such as dithiothreitol (DTT) or 2-mercaptoethanol is also added to cleave protein disulfide bonds and ensure that no quaternary or tertiary protein structure remains. Consequently, when these samples are electrophoresed, proteins separate according to mass alone, with very little effect from compositional differences.

27. (2S,3S)-1,4- bis(sulfanyl)butane-2,3-diol (DTT)

28. Differences Page 28 Native PAGE Separation is based upon charge, size, and shape of macromolecules. Useful for separation and/or purification of mixture of proteins This was the original mode of electrophoresis. SDS PAGE Separation is based upon the molecular weight of proteins. The most common method for determining MW of proteins Very useful for checking purity of protein samples

29. Two gel system  PAGE is widely used to analyze the proteins in complex extracts.  The system actually consists of two gels - a resolving (aka running) gel in which proteins are resolved on the basis of their molecular weights (MWs) and a stacking gel in which proteins are concentrated prior to entering the resolving gel.  Gel matrices are permeated with networks of pores through which the molecules move. The amount of resistance that the matrix presents to the movement of a molecule depends on the diameter of the pore as well as the size and geometry of the molecule

30. Stacking gel: ordering/arranging and conc the macromolecule before entering the field of separation. (4% of acrylamide) pH 6.7 Purpose is to concentrate protein sample in sharp band before enters main separating gel. Running gel: the actual zone of separation of the particle/molecules based on their mobility. (15% of acrylamide) pH 8.9 Pore size: routinely used as 3% to 30% which is of pore size 0.2nm to 0.5nm resp. 30

31. 31

32. Movement of particle [Cl] > [protein-SDS] > [Glycinate]

33. Chemistry of acrylamide polymerization The polyacrylamide gels used to separate proteins are formed by the chemical polymerization of acrylamide and a cross- linking reagent, N,N’methylenebisacrylamide. Polymerization occurs because of free oxygen radicals that react with the vinyl groups in acrylamide and bisacrylamide. The oxygen radicals are generated from the catalyst, ammonium persulfate (APS), when it reacts with a second catalyst, N,N,N’,N’-tetramethylethylenediamine (TEMED).

36. ELECTROPHORESIS INSTRUMENTATION Page 36

37. A typical setup consists of a gel slab sandwiched between two glass plates, with the ends enclosed in upper and lower reservoirs of buffer Samples to be run are loaded in wells at the top of the gel, in conjunction with tracking dye. An electrical voltage is applied between the upper and lower reservoirs, causing the samples to migrate down through the gel.

38. Page 13 Procedure ofSDS- PAGE

39. Procedure Set up Gel Load the Buffer Load Sample Page 39

40. Assembling the glass plates: Assemble the glass plate on a clean surface. Lay the longer glass plate (the one with spacer) down first, then place the shorter glass plate on top of it. Embed them into the casting frame and clamp them properly Make sure that the that the bottom ends of the glass plates are properly aligned. Then place it on the casting stand. Page 40

41. Casting the gels Page 41 2.Prepare 10%ofresolving gel and 4.5% of stacking gel. Prepare the separating gel solution by combining all reagents. Do not add Ammonium persulfate and TEMED. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently. Pour the solution till the mark. (It is ok if you introduce air bubbles, add a layer of isopropanol or distilled water on top of the gel so as to level the poured gel.) Allow the gel to polymerize for 20-30 minutes. Prepare stacking gel. Mix all reagents except APS and TEMED. Drain the isopropanol with strips of filter paper. Add APS and TEMED to the monomer solution (just before pouring) and mix well by swirling gently. (Make sure you keep the comb ready by the side.) Place a comb in the stacking gel sandwich. Allow it to polymerize for 10 minutes.

42. Contents of Gel Page 42

43. Preparation of samples 3.Mix your protein in the ratio 4:1 with the sample buffer. Heat your sample by either: a)Boiling for 5-10 minutes. (works for most proteins ) b)65°C for 10 minutes. c)37°C for 30 minutes. Page 43

44. Running the gel To assemble, take out the gels from the casting frame and clamp them in the gel apparatus. (Make sure that the short plate always faces inside and if you have got only one gel to run use the dummy plate that is available to balance). When the plates are secured, place them in the cassette and then lock it. Place them in the gel running tank. Page 44

45. Fill the inner chamber of the tank with buffer.(Now it is easy to remove the comb, since it is lubricated). Remove the comb CAREFULLY (without breaking the well). [Now the gel is ready to load the samples] Page 45

46. Rinse the loading tip a few times with distilled water. (Make sure that all the water is poured out before loading the samples.) Insert the loading tip to a few mm from the well bottom and deliver the samples into the well. Rinse the syringe with distilled water after loading for a few times. Page 46

47. Attach the power supply by putting the lid (Make sure that the connection is in correct way i.e., black - black and red - red). Set the voltage up to 180 V and run for 1 hour.(Don't allow the dye front to go out of the gel). Page 47

48. Staining the gel After running, switch off the power supply and take out the gel plates, remove the gel. Place the gel in the staining solution for 30 minutes. Page 48

49. Staining solution Page 49 Weigh 0.25g of Coomassie Brilliant Blue R250 in a beaker. Add 90 ml methanol : water (1:1 v/v) and 10ml of Glacial acetic acid,mix properly using a magnetic stirrer. Filter through a Whatman No. 1 filter & store in bottles.

50. Destaining the Gel Destain the gel until the bands are properly seen. Determine the approximate molecular weight of the visualised protein bands by comparing them with the molecular weight ladders(markers). Page 50

51. Destaining solution Page 51 Mix 90 ml methanol: water (1:1 v/v) and 10ml of Glacial acetic acid using a magnetic stirrer and store in appropriate bottles.

52. Staining Coomassie blue-sensitive to 0.1ug of protein Silver- sensitive to 0.002ug of protein, based on ppt of silver ions producing brown stain. greater sensitivity, radioactive samples can be used, allowing for exposure over time to produce images on photographic film, as seen in the sequencing gel on the right To calibrate the relative migrations of molecules of different size, a marker lane is often added, where samples of known size w P i a ll ge 27 migrate to reference positions

53. Page 53

54. Relative Mobility where, Z = charge on the molécule E = Voltage applied and, Rf is measured by: f = frictional resistance Direction of movement is determined fromZ: - if Z 0, then → - if Z = 0, then no movement Page 54

55. Significance of SDS Page 55 SDSisaanionicdetergent(soap)thatcandissolve 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 solubilized by the detergent

56. Advantages of PAGE Page 56

57. Applications Page 57 Used for estimation of molecular weight of proteins and nucleic acids. Determination of subunit structure of proteins. Purification of isolated proteins. Monitoring changes of protein content in body fluids. a)To identify whether a particular protein is pure or not. b)Separation of proteins, prior to Western Blot transfer. c)Species identification. d)Antigen preparation. e)To measure genetic diversity

58. Potential problems with Polyacrylamide gels Page 58 –Under loaded (bands invisible) –Sloppy loading or to little concentration of protein –Bent bands –Tearing –frowning

59. 1. Over loaded 3. Frowning (run too hot) 4. Bent bands. Tearing 2. Tearing 5. Sloppy loading or too low conc. Page 59

60. POLYACRYLAMIDE GELELECTROPHORESIS Page 60

61. References Page 61 BIOCHEMISTRY by Donald Voet & Judith Voet, Wiley publications. BIOCHEMISTRY by Satyanarayana & Chakrapani. Biochemistry by Upadhyay, Upadhyay and Nath. http://amrita.vlab.co.in/?sub=3&brch=186&sim=319&cnt=2 http://www.protocolonline.org/prot/Molecular_Biology/Elec http://www.protocolonline.org/prot/Molecular_Biology/Elec trophoresis/Polyacrylamide_Gel_Electrophoresis PAGE_/in dex.html http://www.sciencedirect.com/science/article/pii/00032697 http://www.sciencedirect.com/science/article/pii/00032697 60900361 http://www.nature.com/nature http://www.nature.com/nature physci/journal/v230/n12/abs/physci230092a0.html https://slideplayer.com/slide/3408337/

62. Protein Gel Electrophoresis 1.Native PAGE 2.Native Gradient PAGE 3.Urea PAGE 4.SDS PAGE 5.SDS Gradient PAGE 6.IEF 7.2D PAGE 8.Western Blot.

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