1. ELECTROPHORESIS 1.Definitions 2.Theory of Electrophoresis 3.Electrophoretic Technique 4.General Procedures 5.Types of Electrophoresis 6.Technical Considerations

2. Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. We will be using agarose gel electrophoresis to determine the presence and size of PCR products. Agarose Gel Electrophoresis

3. 1. DEFINITIONS Electrophoresis –Migration of charged solutes in a liquid medium under an electrical field –Many biological molecules have ionisable groups eg. amino acids, proteins, nucleotides, nucleic acids –Under an electric field -> charged particles migrate to anode (+) or cathode (-)

4. Zone Electrophoresis Migration of charged molecules Support medium –porous eg. CA or agarose –can be dried & kept Same pH & field strength thru’ought Separation based on electrophoretic mobility Separates macromolecular colloids eg. proteins in serum, urine, CSF, erythrocytes; nucleic acids

5. Isotachophoresis Migration of small ions Discontinuous electrolyte system –leading electrolyte (L - ions, high mobility) & –trailing electrolyte (T - ions) Apply sample solution at interphase of L & T Apply electric field -> each type of ion arrange between L and T ions -> discrete zones Separates small anions, cations, organic & amino acids, peptides, nucleotides, nucleosides, proteins

6. Rate of migration depends on: –Net electrical charge of molecule –Size & shape of molecule –Electric field strength –Properties of supporting medium –Temperature of operation

7. 3. ELECTROPHORETIC TECHNIQUE 3a. Instrumentation & Reagents Buffer boxes with buffer plates -> holds buffer Platinum or carbon electrode -> connected to power supply Electrophoresis support -> with wicks to contact buffer Cover -> minimize evaporation (Fig 7-1)

8. 3c. Buffers To carry applied current & to fix the pH => determine electrical charge & extent of ionization => which electrode to migrate Ionic strength of buffer –thickness of ionic cloud -> migration rate -> sharpness of electrophoretic zones –[ion]  -> ionic cloud  -> movement of molecules  Barbital buffers & Tris-boric acid-EDTA buffers

9. 3d. Protein Stains To visualize/locate separated protein fractions Dyes: amount taken up depends on –Type of protein –Degree of denaturation of proteins by fixing agents Types of stains: Table 7-1

10. 4. GENERAL PROCEDURES 4a. Separation Place support material in EP chamber Blot excess buffer from support material Place support in contact with buffer in electrode chamber Apply sample to support

11. cont. Separation Separate component using constant voltage or constant current for length of time Remove support, then -> dry or place in fixative -> treat with dye-fixative -> wash excess dye -> dry (agarose) or put in clearing agent (CA membs)

12. 4b. Detection & Quantitation Express as –% of each fraction present or –absolute concn By densitometry –electrophoretic strip moved past an optical system –absorbance of each fraction displayed on recorder chart

13. 5. TYPES OF ELECTROPHORESIS a.Agarose Gel Electrophoresis b. Cellulose Acetate Electrophoresis c. Polyacrylamide Gel Electrophoresis d. Isoelectric Focusing e. Two-dimensional Electrophoresis

14. 5a. Agarose Gel Electrophoresis (AGE) Use agarose as medium –low concns -> large pore size –higher concns -> small pore size Serum proteins, Hb variants, lactate dehydrogenase, CK isoenzymes, LP fractions Pure agarose - does not have ionizable groups -> no endosmosis

15. Cont. AGE Advantages: –low affinity for proteins –shows clear fractions after drying –low melting temp -> reliquify at 65 o C Disadvantage: –poor elasticity -> not for gel rod system -> horizontal slab gels

16. 5b. Cellulose Acetate Electrophoresis (CAE) Cellulose + acetic anhydride -> CA Has 80% air space -> fill with liquid when soaked in buffer Can be made transparent for densitometry Advantages: –speed of separation –able to store transparent membranes Disadvantages: –presoaking before use –clearing for densitometry

17. cont. CAE Method: –wet CA in EP buffer –load sample about 1/3 way along strip –stretch CA in strips across a bridge –place bridge in EP chamber -> strips dip directly into buffer –after EP, stain, destain, visualise proteins For diagnosis of diseases –change in serum protein profile

18. 5c. Polyacrylamide Gel Electrophoresis (PAGE) Tubular-shaped EP cell -> pour small-pore separation gel -> large-pore spacer gel cast on top -> large-pore monomer solution + ~3ul sample on top of spacer gel Electrophoresis -> all protein ions migrate thru large-pore gels -> concentrate on separation gel -> separation due to retardation of some proteins

19. Average pore size in 7.7% PAGE separation gel about 5nm -> allow most serum proteins to migrate -> impedes migration of large proteins eg fibrinogen,  1 -lipoprotein,  2 - macroglobulin Advantages: –thermostable, transparent, strong, chemically inert –wide range of pore sizes –uncharged -> no endosmosis Disadvantages: –carcinogenic

20. Denaturing PAGE/SDS-PAGE

21. What is SDS-PAGE? Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis A procedure to separate proteins and determine their Molecular Weights.

22. What is so special about SDS? SDS is a negatively charged detergent. Disrupts secondary and tertiary protein structures by breaking hydrogen bonds and unfolding protein. ‘Masks’ charge on protein so that all proteins act the same as regards charge. Prevents protein aggregation. Prevents protein shape from influencing gel run.

25. (i)Denaturing PAGE/SDS-PAGE â Boil sample for 5 mins in sample buffer containing  -mercaptoethanol & SDS â  -mercaptoethanol: reduce disulfide bridges â SDS: binds strongly to & denatures proteins â Proteins denatured -> opens into rod- shaped structures -> separate based on size â Use: –To assess purity of protein –To determine MW of protein

26. (ii) Native PAGE Use non-denaturing conditions -> no SDS or  -mercaptoethanol -> proteins not denatured Proteins separate based on: –different electrophoretic mobilities –sieving effects of gel Use –to obtain native protein/enzyme –to study biological activity

27. Native gradient PAGE example Zavialov et al. Mol. Microbiol. 2002 Native 4-15% gradient PAGE

28. 5d. Isoelectric Focusing To separate amphoteric cpds eg. proteins Proteins moves to zone where: pH medium = pI protein => charge = 0 pI of protein confined in narrow pH range -> sharp protein zones Method: –use horizontal gels on glass/plastic sheets –introduce ampholytes into gel -> create pH gradient

29. cont. IEF Method –apply a potential difference across gel –anode -> area with lowest pH –cathode -> area with highest pH –proteins migrate until it arrives at pH = pI –wash with fixing solution to remove ampholytes –stain, destain, visualise Separations of proteins with 0.01 to 0.02pH unit differences (Fig 7-4)

30. 5e. Two-Dimensional (2D) EP (ISO-DALT) 1st D using IEF EP -> in large-pore medium -> ampholytes to yield pH gradient 2 nd D using molecular weight-dependent EP -> in polyacrylamide -> linear or gradient O’Farrell method: –use  -mercaptoethanol (1 st D) & SDS (2 nd D) Detect proteins using Coomassie dyes, silver stain, radiography, fluorography Separates 1100 spots (autoradiography)

32. Medium pH range (pH 4-7) 116 97 81 66 55 45 30 21 14 kDa pI 4756

33. Narrow pH range (1 pH unit) 5.5 6.05.04.54.0 116 66 97 55 81 30 45 21 14 pI MW (kDa) (4.5-5.5) (4.0-5.0) (5.0-6.0)

34. SDS-PAGE buffers and Solutions Resolving buffer: Stacking buffer: 10% APS: 10X SDS-PAGE Tris-Glycine-SDS running buffer: 3X SDS-PAGE loading buffer: 30% 37.5:1 acrylamide/bisacrylamide solution:

35. Southern blot (DNA) Northern blot (RNA) Western blot (Protein) Eastern blot (???) Immunoblotting

36. IMMUNOASSAYS 1.Basic Concepts & Definitions 2.Measurement of Antibody Affinity 3.Quantitative Methods – competitive & noncompetitive assays

37. 1. BASIC CONCEPTS & DEFINITIONS Immunoassay: use of antibodies to detect analyte 1a. Antibodies Immunoglobulins that bind to Antigens 5 classes: IgG, IgA, IgM, IgD, IgE 1b. Immunogen Protein or a substance coupled to a carrier When introduced into foreign host -> induce Ab to form 1c. Antigen Any material which can react with Ab May not induce Ab formation

38. 1d. Antigen-Antibody Binding Ab molecules have specific binding sites -> bind tightly to Ag -> cause pptn/neutralization/ death Binding of Ag to Ab due to –van der Waals forces –hydrophobic interactions –charged group attractions Can measure Antibody affinity: strength of binding between Ab & Ag

39. 2. MEASUREMENT OF ANTIBODY AFFINITY Binding of Ag to Ab is reversible -> association & dissociation Ag + Ab AgAb Law of mass action: Rate of rxn  to concn of reactants k a [Ag][Ab] = k d [AgAb] K = k a /k d = [AgAb]/ [Ag][Ab] where K is equilibrium constant or affinity constant

40. r/c = nK – rK r = no. of molecules of bound Ag per Ab molecule c = concn of free Ag n = valency of Ab Plot r/c vs r => Scatchard Plot –Straight line with slope k –x intercept gives n –y intercept gives nK K (liters/mole) measures affinity of complex

41. Why measure Affinity of an Antibody? To assess Ab specificity It influences the functional efficiencies of Abs eg. high-affinity Abs are very dependable for various applications: –Diagnostic –Therapeutic –Analytical

42. 3. QUANTITATIVE METHODS Read & Understand from Tietz Fundamentals: –Radial Immunidiffusion Immunoassay –Electroimmunoassay –Turbidimetric & Nephelometric Assays Labeled Immunochemical Assays

43. COMPETITIVE vs NONCOMPETITIVE RXNS A. Competitive Immunoassays Used when have limited reagents (Ag) (i) Simultaneous Competitive Assay Labels Ag (Ag*) & unlabeled Ag compete for binding to Ab The probability of Ab binding to Ag* is inversely  to [Ag] Ab + Ag + Ag* Ab:Ag + A-Ag*

44. (ii) Sequential Competitive Assay Step 1: unlabeled Ag mixed with excess Ab -> binding allowed to reach equilibrium Step 2: Ag* added sequentially -> equilibrate After separation -> det bound Ag* -> calculate [Ag] Larger fraction of Ag bound to Ab than in simultaneous assay If k 1 >> k 2 ->  in Ab:Ag ->  in Ag* binding Provide two- to four- fold improvement in detection limit

45. b. Noncompetitive Immunoassays Used when have excess reagent i.Immobilization of Ab to support –Passively adsorption or bind covalently –Direct or indirect attachment (Table 9-3) ii.Ag allowed to react with Ab -> wash other proteins iii. Add labeled Ab (conjugate) -> reacts with bound Ag iv.Determine bound label -> [Ag*] or its activity is  [Ag]