University:
Boston University
Course:
CAS BI 525 | Biology of Neurodegenerative Diseases
Academic year:

2023
Views:

362

Pages:

19
Author:

Nikolas Trujillo

LDH Purification Process Week 1 Week 2 Week 3* LDH Purification Process Fractions: (bold = containing LDH) 1P 1S 1st cut 2P, 2S 2nd cut 3P, 3S 3P‐D* Wash fractions + NADH eluate Affinity Chromatography • AMP moeity of NADH binds to LDH in the NADH‐binding site ● We are using Affi‐Gel Blue Gel Resin to bind LDH to the column ● NADH: ● ● AMP Crosslinked 4% agarose matrix Agarose beads are linked to Cibacron blue F3GA ligand Cibacron blue F3GA is an analog of AMP (mimics the cofactor of LDH) NADH is later used to elute LDH since NADH binds more tightly than AMP Affinity chromatography & ultrafiltration 3P‐D – dialyzate Activity Assay for total Units & calculate volume to load 5000 U onto column Run affinity column Wash Fractions – Unbound proteins NADH eluate Fractions – purified LDH Assay each fraction for activity & pool fractions with highest activity (≤10 mL) Ultrafiltration (Concentrate to ~1 mL) Purified (concentrated) LDH Affinity chromatography setup i) Preparing the column • Packing the column with resin • Equilibration ii) Sample application & running the column • Binding LDH to the beads • Elution Packing & equilibrating the affinity column Pipet Affi‐gel resin into column Let resin settle, then drain buffer to just above surface. DO NOT let column dry out! Equilibrate column with buffer Drain buffer to just above surface. Column is now ready to load. Loading & running the affinity column Apply dialyzate to column with transfer pipet, then drain to just above surface & collect flow‐ through Wash column with buffer & collect fractions until A280 <0.1 Drain buffer to just above surface. Elute LDH with NADH solution. Collect & assay fractions. Chapter 3F‐G: Procedures overview Part F: I. Column preparation II. Affinity chromatography III. Eluting LDH from column Part G: I. Concentration of purified LDH by ultra‐filtration *NOTE: Some groups might not make it to this step. If you have to wait until the following week, turn in your POOLED LDH to TFs Chapter 3F Procedure Column Preparation *NOTE: This must be started AS SOON AS YOU GET TO LAB. TFs will give pre‐lab talk after column equilibration. 1. Take 5 mL of 50% affinity resin slurry and load column − Your partner should be testing activity for 3P‐dialyzed 2. Allow slurry to settle for a few minutes to correctly pack column − Don’t allow column to dry‐out at any point of the lab!!! 3. Equilibrate with 10‐15 mL of 0.02 M potassium phosphate buffer • If flow rate for column is very slow, contact TF immediately! Chapter 3F Procedure Affinity chromatography: loading the column 1. Measure and record the volume of the 3P‐Dialyzed fraction 2. From calculated activity, determine the volume of 3P‐dialyzed fraction needed for 5000 U • • Don’t forget to take an aliquot for dye‐binding Heart samples: you may need your entire 3P‐D fraction 3. Test the initial flow‐through for activity • Why is this necessary? • What does low activity tell you? • What does high activity tell you? Chapter 3F Procedure Affinity chromatography: washing the column 1. Wash column with 5 mL portions of buffer • Collect washes from column in 1‐2 mL fractions 2. Measure absorbance @ 280 nm of “wash” fractions on the UV‐Vis • • Which macromolecules are absorbing @ 280 nm? What is the significance of A280 < 0.1? Chapter 3F Procedure Eluting LDH from column 1. Begin eluting with 0.2 mM NADH in 0.02 M potassium phosphate buffer • What is in the buffer that allows LDH to elute? • Why does LDH elute from the column? 2. Collect eluate in 1‐2 mL fractions 3. Test eluate fractions for LDH activity 4. Pool the fractions with the highest activity in a 15‐mL conical tube • • Keep total volume below 10 mL Measure and record this volume Tips: Affinity chromatography • When adding liquid to the column, be gentle and do not disturb the resin bed • Never allow any part of the resin/column to dry out! • When column is flowing, one person must be monitoring it at all times • Set up calculation for 5000 U in pre‐lab procedure! • Have TF check before loading sample • If flow rate is very slow, immediately call TFs • Keep flow‐through and all eluate fractions on ice • Why is this important? • Pay attention to NADH concentration for buffers • Why is this important? • Properly label all fraction tubes Chapter 3G Procedure Concentration of purified LDH by ultra‐filtration • Add sample to ultra‐filtration device • Place in ice tray on top of stir plate • Spin internal stir bar slowly • Slowly begin to apply pressure from nitrogen tank • Collect the flow‐through as you concentrate your sample • Collect sample in eppendorf tube when you have ~1.5 – ~1.0 mL of sample left • Place on ice and do not throw your away sample! Tips: Ultra‐filtration concentration • Keep ultra‐filtration device packed in ice in a small plastic box • Assemble unit similar to last week’s Erlenmeyer flask setup • Keep nitrogen pressure low and be patient • No more than one drop of flow‐through every 5 seconds Activity Assays ● Perform LDH Activity Assays for: – 3P dialyzed – “Flow‐through” – Eluate fractions – Ultra‐filtered, purified LDH ● Use aliquots for assays ● Dilute where necessary ● For Week 3, only need 1 dilution for each fraction in ΔA340/min = ‐0.05‐0.25 range What do you use to blank your spectrophotometer? Week 3: Bradford Assays ● Protein Concentration – Bradford (dye‐binding) Assay ● ● Make new standard curve if doing this week Find protein concentration for: – – – – ● ● 3P dialyzed “Flow‐through” Eluate fractions Ultra‐filtered, purified LDH Use aliquots for assays A595 should be within linear region of your standard curve – Dilute protein when necessary What do you use to blank your spectrophotometer? Chapter 3, Week 3 Checklist: At the end of lab, you should have: Prepared four new “sets” of fractions (& recorded volumes of): o 3P‐dialyzed o “Flow‐through” o Eluate fractions o Ultra‐filtered, purified LDH Performed activity assays on all fractions Made a new standard curve & performed Bradford assays (only if had time) Turned in all aliquots and purified LDH to TFs to freeze Purification Table: Correction Factors • Add correction factors where appropriate & carry through subsequent steps *Note: Example table on page 95 contains errors! (Do not follow.) Fraction Volume (mL) Volume taken for next step (mL) Corrected volume (mL) Homogenate 170 60 (used for spin) ‐ 1S 45 all (170/60) x 45 = 128 1st salt cut 65 62 (used for spin) ‐ 2S 55 all (170/60)(65/62) x 55 = 163 2P 22 ‐ (170/60)(65/62) x 22 = 65 2nd salt cut 57 50 (used for spin) ‐ 3S 30 ‐ (170/60)(65/62)(57/50) x 30 = 102 3P 20 all (170/60)(65/62)(57/50) x 20 = 68 3P‐D 22 15 (loaded column) (170/60)(65/62)(57/50) x 22 = 75 Pooled 7 all (170/60)(65/62)(57/50)(22/15) x 7 = 35 Conc’d LDH 1 ‐ (170/60)(65/62)(57/50)(22/15) x 1 = 5

LDH Purification Process

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