University:
Boston University
Course:
CAS CH 102 | General Chemistry 2
Academic year:

2021
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309

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17
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pudlejern7cjc

Purification of Lactate Dehydrogenase (LDH) Purpose: Understand to how to purify proteins via affinity columns Learn how to pack and equilibrate a column Use ultrafiltration to concentrate protein samples Affinity Chromatography ● ● Technique for separating proteins based on binding properties of a macromolecule to its ligand Possible Ligands: ● ● ● ● ● ● Molecules that bind the ligand will be absorbed on to the column ● ● Cofactors Allosteric effectors Substrate analogs Nucleic acids Anything that the protein will easily recognize and bind, but other proteins will not bind Highly specific purification method Molecules that do not bind the ligand will be washed off ● Prevents non-specific binding Affinity Chromatography ● ● We are using Affi-Gel Blue Gel Resin (Bio-Rad) Contains: ● ● ● Crosslinked 4% agarose matrix Linker molecule to attach agarose to Cibarcon blue F3GA Cibarcon blue F3GA dye that mimics AMP in LDH Purification • AMP – Nucleotide that interacts with LDH in NADH binding site • This site is a general nucleotide binding site • Other dehydrogenases and proteins that bind AMP will also bind to this site • NADH is used to elute LDH since NADH binding more tightly to site than AMP How to Pack and Load a Column in Biochemistry Pipet in all resin Equilibrate on side of column with column, open buffer stopcock, let settle, but DO NOT let column dry out! Drain Buffer Apply sample Let sample drain Add buffer to to surface, with pipet to to surface, close top, open stop close side of stopcock cock, collect stopcock column, open fractions stopcock Ultrafiltration ● ● ● ● ● Technique for concentrating protein samples Uses membrane with molecular weight cutoff below protein interest Removes water, salts, and small impurities Nitrogen gas is used as pressure to push water out Stirring allows protein concentration to remain equal throughout sample Activity Assay for total number of units! Load 5,000 Units on to column 3P–Dialyzed Affinity Purification Wash Fractions – Nonspecifically bound proteins Flow Chart for LDH Purification Wash fractions with KPO4 Buffer Elute fractions with KPO4/NADH NADH Eluted Fractions Buffer – Specifically bound to resin Ultrafiltration – removes buffer Concentrate samples to ~1 ml Purified LDH Pool fractions with highest activity Procedure ● Assay 3P-Dialyzed Fraction ● Affinity Chromatography ● Ultrafiltration ● Activity Assays and Dye-Binding on all necessary fractions Keep everything on ice! Especially LDH extract! Procedure ● Assay 3P-Dialyzed Fraction ● Calculate how many total units you have in the 3P-Dialyzed Fraction: [Activity] = ΔC = (ΔA340/min)/(εapp in mM) [Activity] = ΔC = (0.12/min)/(6.21 µmol/ml) = 0.019 units/ml [Activityundiluted] = (ΔC units/ml)(Total Volume)(Dilution Factor) (Volume of enzyme used) [Activityundiluted] = (0.019 units/ml)(3 ml)(400) = 464 units/ml (0.05 ml) Total Activity in 3P Dialyzed = (Activity 3P-D)(Total Volume 3P-D) Total Activity in 3P-Dialyzed = (464 units/ml)(15 ml) = 6957 total units Procedure ● Assay 3P Dialyzed Fraction ● Calculate volume of 3P-Dialyzed Fraction needed for 5000 Units: Volume for 5000 units = (5000 units)(Total volume 3P-D)/(Total Units 3P-D) Volume for 5000 units = (5000 units*15 ml) = 10.8 ml 6957 units ● Load 10.8 ml on to affinity column ● If you have > 5000 units, load all but 250 μl aliquot Procedure ● Affinity Chromatography ● Pour 5 ml of affinity resin in small column with stopcock ● Let resin buffer drip until settled DO NOT DRY COLUMN! ● Wash resin with 10-15 ml 0.02 M KPO4 Buffer ● Load 5000 units of 3P-Dialyzed on to column ● – Save at least 250 µl aliquot for dye-binding – Be careful not to disturb surface of resin Allow flow through to pass through column – Collect all in one fraction – test activity Procedure ● Affinity Chromatography ● ● Wash with KPO4 Buffer – Collect 1-2 ml fractions – Bring to TF’s to check Abs 280 nm in UV spectrophotometer – Stop washing when Abs 280 nm < 0.1 Elute fractions with NADH/KPO4 Buffer What proteins come off in this step? – Collect about 10-15 fractions (1-2 ml each) – Assay fractions for enzyme activity and protein content – Pool up to 10 ml with highest activity – Take small aliquot (~250 μl) of pooled fractions for activity and dye binding Why does LDH elute here? Procedure ● Ultrafiltration ● ● ● ● ● Bring pooled fractions to TF at ultrafiltration setup Add sample to ultrafiltration cell on stir plate with ice bucket Attach nitrogen line and stirring to concentrate sample to ~ 1 ml Carefully remove sample without breaking membrane Do activity assays and dye binding on concentrated sample Procedure ● Activity Assays ● Do LDH Activity Assays on: – 3P-Dialyzed (before loading column) – Flow through – buffer passing through column during sample loading (1 combined fraction) – Activity of NADH eluted fractions – find peak and test ALL POOLED FRACTIONS – Pooled Affinity Fraction – Pooled Concentrated Fraction ● Use 1 ml aliquots for assays ● All assays need to be in range of ΔA340/min of 0.05-0.25 What do you use to blank your spectrophotometer? Procedure ● Protein Concentration – Dye Binding Assay ● ● ● Find protein concentration for: – 3P-Dialyzed – Pooled Affinity Fraction – Pooled Concentrated Fraction – Anything you did not finish in weeks 1 or 2 Use 1 ml aliquots for protein concentration A595 should be within linear region of your standard curve – Dilute protein when necessary What do you use to blank your spectrophotometer? Lab Notebook – Chapter 3 ● ● ● Purification details – weights/volumes of each step Apparent extinction coefficient and spectra LDH activity assays – ● ● ● ● ● ● ● ● A340 vs. Time for 1 assay graph ΔA340/min vs. [Enzyme] for dilutions of 1S (graph) Rates, Activity, and Total Activity calculations for all fractions Affinity purification – assays of fractions, graph of activity vs. fraction Dye-Binding standard curve, results table, and calculations Purification table and sample calculations All questions in the notebook section 1 discussion/conclusion for entire lab Purification Table Calculations Specific Activity (units/ ΔA340/ Dilution mg) Yield (%) min Factor Corrected Activity Total Total Volume Volume (units/ Protein Activity Protein Fraction (mL) (mL) mL) (mg/mL) (units) (mg) 160 160 Homogenate 1S 80 160.0 272.5 125 43594 20000 2.2 100.0 0.071 400 2S 30 60.0 456.0 30 27362 1800 15.2 62.8 0.118 400 2P 15 30.0 10.0 44 301 1320 0.2 0.7 0.208 5 3S 35 70.0 12.0 17 839 1190 0.7 1.9 0.248 5 3P 17 34.0 560.4 26 19053 884 21.6 43.7 0.145 400 3P-D 18 36.0 510.1 22 18365 792 23.2 42.1 0.132 400 Pooled 8 32.0 499.7 4 15991 128 124.9 36.7 0.129 400 Conc. 2 8.0 1959.4 15 15675 120 130.6 36.0 0.169 1200 Only used 80 ml of original homogenate, therefore everything is multiplied by 2 Only loaded 9 ml for 5000 units for affinity column, therefore volume is multiplied by 4 (8 ml*4 = 32 ml) Final Example Purification Table Corrected Volume Fraction (mL) 160 Homogenate 1S 160.0 2S 60.0 2P 30.0 3S 70.0 3P 34.0 3P-D 36.0 Pooled 32.0 Conc. 8.0 Activity (units/mL) Protein (mg/mL) Total Activity (units) 272.5 456.0 10.0 12.0 560.4 510.1 499.7 1959.4 125 30 44 17 26 22 4 15 43594 27362 301 839 19053 18365 15991 15675 Total Protein (mg) Specific Activity (units/mg) Yield (%) 20000 1800 1320 1190 884 792 128 120 2.2 15.2 0.2 0.7 21.6 23.2 124.9 130.6 100.0 62.8 0.7 1.9 43.7 42.1 36.7 36.0

Purification of Lactate Dehydrogenase

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