Chapter 5: Structural Characteriza1on of LDH. Chapter 5: Overview

Transcription

1 Chapter 5: Structural Characterizaon of LDH Purpose: Learn how to: ) Use gel filtra3on chromatography 2) Determine na3ve molecular weight of proteins 3) Assay a mix of different standard proteins Chapter 5: Overview Week : Gel Filtraon Chromatography Separate LDH from standard proteins by na3ve size Determine Na3ve MW Week 2: Denaturing Polyacrylamide Gel Electrophoresis (SDS- PAGE) Separate LDH from other proteins by subunit size Determine Subunit MW Week 3: Nave Electrophoresis (Zymograms) Confirm the quaternary structure of LDH from Weeks & 2

2 Gel Filtra3on Chromatography Size- Exclusion Chromatography Technique to separate molecules by size Resin: Gel Matrix with par3cles or beads of a par3cular size with small pores Can be used for prepara3ve or analy3cal separa3ons Gel Filtra3on Chromatography 2

3 Types of Gel Filtra3on Resins Cross- linked Dextran Sephadex Cross- linked Agarose Bio- Gel A, Sepharose, Superose Cross- linked Polyacrylamide Bio- Gel P Combinaon Dextran with Polyacrylamide Sephacryl Dextran with Agarose Superdex We are using Sephadex G- 50 Dry resin has pores with a maximum size of 50,000 Da Resin will swell in water and aqueous salt solu3ons Can frac3onate proteins from 5, ,000 Da Determining Molecular Weight from Gel Filtra3on 8 7 Linear Relaonship between Log MW and V e /V o V e = Elu3on Volume of Compound V o = Void Volume of Column, volume of very large material that does not bind to any of the resin Log MW y = -.06x R² = 0.97 Ferri3n Catalase Ovalbumin LDH Eluon Point Calculate MW from equa3on for graph Myoglobin Ve/Vo 3

4 Column Packing and Running Review Pipet in all resin on side of column, open stopcock, let seble, but DO NOT let column dry out! Equilibrate column with buffer using buffer reservoir Drain Buffer to surface, close stopcock Apply sample with pipet to side of column, open stopcock See p. 29 for more diagrams Let sample drain to surface, close stopcock Add buffer to top, abach buffer reservoir, open stop cock, collect with fracon collector Procedure: Chapter 5- Week TF s will pack and equilibrate column for you We will use buffer reservoirs and frac3on collectors for this lab Using your final concentrated LDH sample, calculate volume needed to load units (2000 units/ ml) = (50 units/x ml) x = ml, 25 μl Add LDH to Calibra3on 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 separaon and possibly lose all your protein! 4

5 Procedure: Chapter 5- Week Aher protein almost completely loaded, add buffer to top and abach buffer reservoir Collect frac3ons with frac3on collector About 25 drops/frac3on (~0.5 ml/frac3on, measure exact volume) As calibra3on proteins move through column they will separate Ferrin Light yellow (MW = 440,000) Catalase (MW = 220,000) LDH Ovalbumin (MW = 45,000) Myoglobin Light brown (MW 7,300) Dilute each frac3on with 2.7 ml, 0.02 M KPO 4 Buffer Procedure: Chapter 5- Week Start Analyzing Column Frac3ons Find Peaks! Ferrin A 360 in your spectrophotometer Peak of ferri3n is your V o Catalase Assay with KMnO 4 Time sensi3ve assay, # of aliquots = catalase ac3vity LDH Ac3vity Assay (ΔA 340 /min) Use 50 µl of diluted frac3ons for assay Ovalbumin A 280 in UV- spectrophotometer Myoglobin A 40 in your spectrophotometer 5

6 Procedure: Chapter 5- Week First: Find Ferrin and Myoglobin Next: LDH Ac3vity Assay, then Ovalbumin Last: Catalase Assay Start at ferr3n peak and assay to LDH peak 50 µl of diluted frac3on ml H 2 O 2, mix, 6 min incuba3on, quench with 2.0 ml H 2 SO 4 Add 00 µl aliquots of KMnO 4 un3l reac3on stays pink Fewest aliquots of KMnO 4 = Highest Ac3vity of Catalase Need to invert catalase values for graphs Procedure: Chapter 5- Week Normalize All Peaks to.0 and Graph on Axis Normalized Acvity Fracon Number Ferri3n Catalase Determine Na3ve MW from V e /V o vs Log MW graph LDH Ovalbumin Myoglobin 6

7 Determining Molecular Weight from Gel Filtra3on 8 7 Linear Relaonship between Log MW and V e /V o V e = Elu3on Volume of Compound V o = Void Volume of Column, volume of very large material that does not bind to any of the resin Log MW y = -.06x R² = 0.97 Ferri3n Catalase Ovalbumin LDH Eluon Point Calculate MW from equa3on for graph Myoglobin Ve/Vo 7