University:
Boston University
Course:
CAS CH 101 | General Chemistry 1
Academic year:

2022
Views:

44

Pages:

12
Author:

leptommoxi

MONITORING ENYME PURIFICATION Enzyme Purification Step Crude Extract (1S) (2S) (2P) (3S) (3P) (3PD) (3PD-FT) (3PD-W) (Emax) (E pooled) (E-concentrated) Volume (ml) Activity (units/ml) [protein] (mg/ml) Total Activity (units) Total Protein (mg) Specific Activity (units/mg) Yield % LDH PURIFICATION FLOWCHART Bovine Muscle or Heart Tissue Corresponding general protein purification step Mince/ blend 4X 15 sec high pulse Centrifuge 15 min @14,500 rpm 1.Cell Lysis (Homogenization) 2. Separation of soluble and insoluble components of the lysed cells (Centrifugation) 1P- precipitate 1S- supernatant (cell debris, nuclei) (crude extract- starting material) Add saturated (NH4SO4 (aq) to 40% saturation centrifuge 10 min @14,000 rpm 2P- precipitate 2S- supernatant 3P- precipitate 3S- supernatant Redissolve in 15 ml KPO4 Dialyze 3. Separation of desired protein from other proteins (fractionation) by salting out (Ammonium sulfate precipitation) 4. Replacement/exchange of protein environment (dialysis) 3PD- dialyzed 5. Separation of desired protein from other proteins (fractionation) by column chromatography WK3 (Affinity Chromatography) Perform Affinity Chromatography FT- flow through W- wash E- eluate Pool and concentrate PURIFIED MUSCLE or HEART LDH MODULE 3C What will you do? ● ● ● Run the dialyzed 3PD on an affinity chromatography column Determine which fractions have LDH activity Pool them together and concentrate Why is the procedure calling for a centrifugation step for your dialyzed sample before applying it to the column? AFFI_GEL AFFINITY CHROMATOGRAPHY Why does it work? Dinucleoside diphosphate Cibacron blue F3GA dye coupled to agarose Analog of AMP and binds to LDH at the NADH binding site (Sulphates resemble Binds to LDH at the phosphate and ring resembles ribose) NADH binding site Absorbs visible light appearing dark blue NADH has a higher binding affinity to LDH than AMP NADH BINDING POCKET OF LDH COLUMN PREPARATION Pipet 6 mls of 50% slurry Pipet 1015 mls of 20mM KPO4 If column flow rate is very slow when you are draining the equilibration buffer, do NOT add your sample, call your TF MODULE 3C- Affinity Chromatography Loading MAKE SURE TO KEEP ALL THE FRACTIONS ON ICE AT ALL TIMES • Retrieve your dialyzed 3P sample from the dialysis bag (3PD). Transfer to a pre-chilled 50 ml tube and spin the sample @ 14,500 rpm to remove any precipitation (if needed). • Take two 250 µl aliquots, freeze one and keep the other on ice for Bradford and LDH assay • Measure the volume of the remaining 3PD and load ALL of it onto the affinity column. • Mix the sample with the resin in the column and let the sample bind the resin for 10 min (batch binding) • Collect flow-through (3P-FT)– take two 250 µl aliquots, freeze one and keep the other on ice for assays. • Check enzyme activity for the loaded sample (3PD) and the flowthrough (3PD-FT). You should see a significant difference in the measured activity. If you do NOT see a difference, consult you TFs MODULE 3C- Affinity Chromatography Washing • Apply 30 ml of wash buffer (20 mM K2PO4) to the column and collect the wash into a 50 ml tube (3PD-W). • Add 1 more ml of wash buffer and collect it this additional wash into a UV-Vis cuvette • Check its absorbance using Cary 60s. • If A280<0.1 continue with elutions. If not wash with 10 more mls into the same 50 ml tube and recheck A280 MODULE 3C- Affinity Chromatography Elution • Close the outlet of your column and add 1.5 mls of elution buffer (0.2 mM NADH in 0.02 M K2PO4/100 mM NaCl) to the column. • Let it sink in for 1-2 minutes and collect the eluate (3PD-E1). • Keep adding more elution buffer in 1.5 ml portions and collecting elution fractions. • After E4, close the column outlet, add the next 1.5 ml of elution buffer, stir the resin, and wait 2 minutes before opening the outlet and continue collecting. • Check activity of E1-E4 using 1:50 and 1:500 dilutions. Then continue checking the odd numbered elutions up to E9. Call your TF if you still have considerable activity in E9. Pay attention to buffer labels. ELUTION BUFFER HAS NADH MODULE 3C- Affinity Chromatography Pooling Elution Fractions • Identify the highest activity fraction. Take two 50 µl aliquots of this highest activity fraction, freeze one and keep the other on ice for assays. • Pool the rest of the fractions that contained significant enzyme activity (consult your TFs if you are not sure which ones to pool) • Take two 250 µl aliquots of the pooled fractions. freeze one and keep the other on ice for assays. • Measure the exact volume of the remaining pooled elutions before concentration. CONCENTRATING POOLED ELUTIONS • Obtain a pre-equilibrated concentrator (Vivaspin15) MWCO 30,000 Da from your TFs • Pipet in the pooled LDH elutions • Spin @3,500 g, 4C for 8-10 minutes • Transfer the concentrated sample into a pre-chilled 1.5 ml tube and record its exact volume. • If you see precipitation, spin down the sample on a benchtop centrifuge and then take samples for assay from the supernatant and freeze the rest of the supernatant. 15 mls  1-5 ml THIS IS YOUR PRECIOUS PURE LDH DATA TO FOCUS ON THIS WEEK Enzyme Purification Step Crude Extract (1S) (2S) (2P) (3S) (3P) (3PD) (3PD-FT) (3PD-W) (Emax) (E pooled) (E-concentrated) Volume (ml) Activity (units/ml) [protein] (mg/ml) Total Activity (units) Total Protein (mg) Specific Activity (units/mg) Yield %