University:
Boston University
Course:
CAS CH 214 | Organic Chemistry with Qualitative Analysis
Academic year:

2022
Views:

113

Pages:

14
Author:

paproszekmca5

Structural Characterization of LDH Purpose: Learn how to: 1) Pour an SDS Polyacrylamide gel 2) Prepare samples for SDS-PAGE 3) Determine purity and subunit MW of your LDH Overview ● ● ● Week 1: Gel Filtration Chromatography ● Separate LDH from standard proteins by native size ● Determine Native MW Week 2: Denaturing Polyacrylamide Gel Electrophoresis (SDS-PAGE) ● Separate LDH from other proteins by subunit size ● Determine Subunit MW Week 3: Native Electrophoresis (Zymograms) ● Confirm the quaternary structure of LDH from Weeks 1 & 2 Separating Proteins by Electrophoresis ● ● Applied electric field separates proteins ● Native Electrophoresis ● Many factors effect rate: ● Isoelectric points ● Titration curves ● ● ● ● Molecular weight ● ● Maintains tertiary and quaternary structure Denaturing Electrophoresis ● Hydrodynamic properties Separation of full protein by size and charge Denature of protein into subunits Separation of those denatured subunits by size 2D Electrophoresis ● ● Separation of protein by pI in first direction Separation by size in second direction Polyacrylamide Gels ● ● ● ● ● Matrix for separation Acts as homogeneous support to prevent diffusion of proteins out of gel Acts as molecular sieve slowing the migration of proteins in proportion to their charge-to-mass ratio Concentration can be varied to separate wide range of proteins Speed of separation is related to electrophoretic mobility Polyacrylamide Gel Formation Need to remove any un-polymerized monomer – it can react with protein functional groups! Denaturing Polyacrylamide Gel Electrophoresis – SDS-PAGE ● ● Method to separate proteins by molecular weight of denatured subunits Treat protein with strong denaturant (SDS) and sulfhydryl compound (βME or DTT) ● ● ● ● Unfolds polypeptide chains into random coils Coats protein surface to give uniform negative charge Prevents disulfide bonds from forming between subunits Denatured subunits can be permanently separated with SDS containing buffers ● ● Large excess of SDS SDS binds to any protein at 1.4 g SDS/g of protein ● Gives all proteins same charge/mass ratio SDS-PAGE ● Discontinuous (Disc) electrophoresis ● ● ● ● Layers of gel for better separation based on pH changes (p. 165-8) ● Different acrylamide percentages in gel affect separation Running Gel – bottom layer, high pH (8.8), higher % acrylamide Stacking Gel – top layer, lower ● Higher % gel – better separation of small proteins pH (6.8), lower % acrylamide, where the wells are located ● Lower % gel – better separation of large proteins Stacking layer for loading samples & stacking condition, running layer for separation Relative Mobility Linear relationship between mobility of a protein and Log MW •Measure distance from top of gel to solvent front •Measure distance from top of gel to protein band interest Rm = Distance to protein band on interest/ Distance to Solvent Front 5.5 200000 116250 97400 Log MW 5 66200 4.5 45000 31000 4 21500 y = -1.2372x + 5.303 R² = 0.9972 3.5 14400 Solvent Front 3 0 0.2 0.4 0.6 Relative Mobility (Rm) 0.8 1 Protein Standards used in Lab 5 on p. 156 of Lab Manual Procedure: ● Prepare Samples ● Assemble Gel Apparatus ● Load Samples ● Run Gel ● Pour Gels (for the next lab section) ● Staining and Visualization Procedure: ● Prepare Samples ● Crude Extract – 30-50 μg 3P-Dialyzed – 15-30 μg ● Purified LDH – 2-5 μg ● ● ● Calculate volumes before you come to the lab! SDS-PAGE Standards – 5 μl aliquot, ready to load, 1 per gel Total Sample Volume = 20 μl ● ● ● ● Use concentration from DyeBinding from Chapter 3! 2 μl 10X Sample Buffer (thick blue liquid) Up to 18 μl of sample to put at the appropriate concentration of protein Water to bring total volume to 20 μl Denature samples 1-2 min at ~ 90°C Procedure: ● Assemble Gel Apparatus: ● ● ● Load Samples: ● Use gel loading tips Yellow loading guides can help line up wells ● Write down your loading scheme! ● ● 2 groups/gel, 2 gels/gel box Pictures p. 135-136 and demo! Run Gel: ● 50 V until through the stacking gel, 200V after that ● Run until blue tracking dye reaches bottom of gel ● Total time ~ 1 hr Procedure: ● Pour Gels: ● Put together short plate and spacer plate (1.5 mm) ● Lock together with casting clamp and stand (pictures p. 133) ● Check plates for leaks with water ● Prepare Running Gel – Recipe p. 134 (100 µl APS) ● ● – Pour or pipet into plate, save extra and see when it polymerizes – Overlay with isobutanol Prepare Stacking Gel – Recipe p. 134 (40 µl APS) – Pour or pipet into plate, save extra and see when it polymerizes – Insert comb to form wells Put poured gel in buffer at 4°C for the next section Procedure: ● Staining and Visualization: ● Remove gel from plates ● Nick a corner so you know the orientation ● Stain with Coomassie Brilliant Blue R-250 ● Destain with 1:5:5 Acetic Acid/Methanol/Water solution ● Image on gel doc ● ● Calculate Rm values for standards and unknown protein bands Make graph of Log MW vs Rm Calculate Rm Values Make Plot of Log MW vs. Rm Linear relationship between mobility of a protein and Log MW •Measure distance from top of gel to solvent front •Measure distance from top of gel to protein band interest Rm = Distance to protein band on interest/ Distance to Solvent Front 5.5 200000 116250 97400 Log MW 5 66200 4.5 45000 31000 4 21500 y = -1.2372x + 5.303 R² = 0.9972 3.5 14400 Solvent Front 3 0 0.2 0.4 0.6 Relative Mobility (Rm) 0.8 1