Polyacrylamide Gel Electrophoresis of Proteins and the Enzyme-Linked Immunosorbent Assay 1

 Introduction to the theory & application of electrophoresis.  Use SDS-PAGE to separate proteins in crude spinach leaf extracts & NH 4 SO 4 fractions from lab 6.  Observe large & small subunits of Rubisco (55 & 14 kD).  Estimate/verify the MW’s of BSA vs. Lysozyme (67 & 14 kD).  Introduction to the theory & application of ELISA (Enzyme- Linked Immuno Sorbent Assays).  Perform a simple (hypothetical) pregnancy test (simulated detection of human chorionic gonadotropin hormone). 2

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 Power Supply – source of direct current; provides electromotive force (voltage) to drive migration of charged solute molecules.  Gel – a stationary matrix that provides differential physical resistance to migration of charged solute molecules.  Buffer – controls pH; dissolved ions help conduct electricity.  Electrodes – conduct / deliver current into buffer & gel.  Gel Box / Container – holds gel & buffer in place; insulates against short-circuits. 4

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N,N’-methylene- bis-acrylamide Acrylamide Monomers Free Radicals Cross-Linked Polyacrylamide 8 Ammonium Persulfate

Polyacrylamide9Agarose

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Where:V = velocity of charged molecule migration. E = potential difference (voltage) of electric field. q = charge of the molecule. f = frictional coefficient (physical resistance). f = frictional coefficient (physical resistance).Thus:  Molecules with a greater charge will move faster.  Gel resistance tends to slow the velocity of migration.  Larger molecules experience greater resistance, and thus migrate slower than smaller molecules. V = E x qE x qE x qE x qf 11

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 Protein samples are treated with a mixture of SDS,  -mercaptoethanol at high temperatures (90 – 95 °C).  SDS destabilizes hydrophobic interactions.   -ME reduces disulfide bridges.  SDS binds to linear polypeptide at a ration of  1.4 g SDS/g protein – gives proteins a constant charge:mass ratio (i.e. charge is directly  MW).  Protein migration in gel is inversely  MW (smaller proteins migrate farther than larger proteins). Sodium Dodecyl Sulfate  -mercaptoethanol Potent Denaturation Agent (Subunits dissociate, polypeptides unfold) 15

16 If an unknown protein moves with an R f of 0.5, log 10 of its MW = Thus, MW = = 41,687.

 Prepare lab 6 samples for electrophoresis (add glycerol, SDS,  ME & heat).  Need 2 mg/mL  Add “2X” treatment buffer  Assemble electrophoresis apparatus.  Load samples onto gel & run.  Perform ELISA pregnancy test simulation.  Stain/destain gels.  TA’s take photographs. 17

18 Protein ladder BSA/lyzozyme Cell-pellet Cell supernatant ( very light band) Cell-free extract

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 An enzyme-antibody conjugate that still recognizes it’s antigen and is able to carry out a colorimetric reaction.  Antibody (with attached enzyme) binds to antigen.  Colorless reactants are provided; colored products are produced.  Location & intensity of color reaction indicates antigen detection. 20

 Typically performed in 96-well polystyrene micro- titer plates.  Polystyrene tightly adsorbs many biomolecules, especially proteins.  After antigen binding, wells are rinsed and treated with a series of reagents to detect antigen. 21

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