University:
Boston University
Course:
CAS CH 524 | Chemical Biology Laboratory
Academic year:

2022
Views:

447

Pages:

13
Author:

Melina Hobbs

Structural Characterization of LDH Purpose: Learn how to: 1) Use gel filtration chromatography 2) Determine native molecular weight of proteins 3) Assay a mix of different standard proteins 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 Gel Filtration Chromatography ● ● ● ● Size-Exclusion Chromatography Technique to separate molecules by size Resin: Gel Matrix with particles or beads of a particular size with small pores Can be used for preparative or analytical separations Gel Filtration Chromatography Types of Gel Filtration Resins ● ● Cross-linked Dextran – Sephadex Cross-linked Agarose –Bio-Gel A, Sepharose, Superose ● Cross-linked Polyacrylamide – Bio-Gel P ● Combination – ● ● ● Dextran with Polyacrylamide – Sephacryl Dextran with Agarose – Superdex We are using Sephadex G-150 ● ● ● Dry resin has pores with a maximum size of 150,000 Da Resin will swell in water and aqueous salt solutions Can fractionate proteins from 5,000-300,000 Da Determining Molecular Weight from Gel Filtration Linear Relationship between Log MW and Ve/Vo Ve = Elution Volume of Compound Vo = Void Volume of Column, volume of very large material that does not bind to any of the resin 8 7 Log MW 6 Catalase Ferritin 5 Ovalbumin 4 Myoglobin 3 LDH Elution Point Calculate MW from equation for graph y = -1.06x + 6.77 R² = 0.97 2 1 0 0 0.5 1 1.5 Ve/Vo 2 2.5 Column Packing and Running Review Pipet in all resin Equilibrate on side of column with column, open buffer using stopcock, let buffer settle, but DO reservoir NOT let column dry out! Drain Buffer Apply sample Let sample to surface, with pipet to drain to close side of surface, close stopcock column, open stopcock Add buffer to top, stopcock attach buffer reservoir, open stop cock, collect with fraction collector Procedure: Chapter 5-Week 1 ● ● ● TF’s will pack and equilibrate column for you We will use buffer reservoirs and fraction collectors for this lab Using your final concentrated LDH sample, calculate volume needed to load 50-100 units (2000 units/1 mL) = (50 units/x mL) x = 0.025 mL, 25 μL ● ● Add LDH to Calibration Proteins – total volume ~ 0.5 mL Drain buffer in column to surface and load protein down side of column Do not dry column! You will have very poor separation and possibly lose all your protein! Procedure: Chapter 5-Week 1 ● ● After protein almost completely loaded, add buffer to top and attach buffer reservoir Collect fractions with fraction collector ● ● As calibration proteins move through column they will separate ● ● ● ● ● ● About 25 drops/fraction (~0.5 mL/fraction, measure exact volume) Ferritin – Light yellow (MW = 440,000) Catalase – (MW = 220,000) LDH Ovalbumin – (MW = 45,000) Myoglobin – Light brown (MW – 17,300) Dilute each fraction with 2.7 mL, 0.02 M KPO4 Buffer Procedure: Chapter 5-Week 1 ● Start Analyzing Column Fractions – Find Peaks! ● Ferritin – A360 in your spectrophotometer – ● Catalase – Assay with KMnO4 – ● Peak of ferritin is your Vo Time sensitive assay,  # of aliquots =  catalase activity LDH – Activity Assay (ΔA340/min) – Use 50 µl of diluted fractions for assay ● Ovalbumin – A280 in UV-spectrophotometer ● Myoglobin – A410 in your spectrophotometer Procedure: Chapter 5-Week 1 ● First: Find Ferritin and Myoglobin ● Next: LDH Activity Assay, then Ovalbumin ● Last: Catalase Assay – Start at ferrtin peak and assay to LDH peak ● ● ● 50 µL of diluted fraction + 4.0 mL H2O2, mix, 6 min incubation, quench with 2.0 mL H2SO4 Add 100 µL aliquots of KMnO4 until reaction stays pink Fewest aliquots of KMnO4 = Highest Activity of Catalase – Need to invert catalase values for graphs Procedure: Chapter 5-Week 1 ● Normalize All Peaks to 1.0 and Graph on 1 Axis 1 0.9 Normalized Activity 0.8 0.7 0.6 Ferritin 0.5 Catalase 0.4 LDH Ovalbumin 0.3 Myoglobin 0.2 0.1 0 0 5 10 15 20 25 30 Fraction Number ● Determine Native MW from Ve/Vo vs Log MW graph Determining Molecular Weight from Gel Filtration Linear Relationship between Log MW and Ve/Vo Ve = Elution Volume of Compound Vo = Void Volume of Column, volume of very large material that does not bind to any of the resin 8 7 Log MW 6 Catalase Ferritin 5 Ovalbumin 4 Myoglobin 3 LDH Elution Point Calculate MW from equation for graph y = -1.06x + 6.77 R² = 0.97 2 1 0 0 0.5 1 1.5 Ve/Vo 2 2.5