Announcements. Next week s discussion will have a quiz on Chapter 3fg and Chapter 11ab Computer Lab (Chapter 11ab): 10/17 10/22

Transcription

1 Announcements Next week s discussion will have a quiz on Chapter 3fg and Chapter 11ab Computer Lab (Chapter 11ab): 10/17 10/22 SCI 162 will be open for 2 hours of each lab section to finish Chapter 3 Chapters 3 and 11 due during Lab 4 (10/24 10/29)

2 Chapter 3 Week 3 Parts F,G 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

3 Affinity Chromatography Technique for separating proteins based on binding properties of a macromolecule to its ligand Possible Ligands: Cofactors Allosteric effectors Substrate analogs Nucleic acids Anything that the protein will easily recognize and bind, but other proteins will not bind Molecules that bind the ligand will be absorbed on to the column Highly specific purification method Molecules that do not bind the ligand will be washed off Prevents non-specific binding

4 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

5 How to Pack and Load a Column in Biochemistry Pipet in all resin on side of column, open stopcock, let settle, but DO NOT let column dry out! Equilibrate column with buffer Drain Buffer to surface, close stopcock Apply sample with pipet to side of column, open stopcock Let sample drain to surface, close stopcock Add buffer to top, open stop cock, collect fractions

6 Technique for concentrating protein samples Ultrafiltration 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

7 Activity Assay for total number of units! Load 5,000 Units on to column Wash Fractions Nonspecifically bound proteins 3P Dialyzed Affinity Purification Flow Chart for LDH NADH Eluted Fractions Specifically bound to resin Ultrafiltration removes buffer Purification Wash fractions with KPO 4 Buffer Elute fractions with KPO 4 /NADH Buffer Pool fractions with highest activity See flow chart p. 70 Concentrate samples to ~1 ml Purified LDH

8 Week 3: Procedure Assay 3P-Dialyzed Fraction Affinity Chromatography Ultrafiltration Activity Assays and Dye-Binding on all necessary fractions Keep everything on ice! Especially LDH extract!

9 Week 3: Procedure Assay 3P-Dialyzed Fraction Calculate how many total units you have in the 3P-Dialyzed Fraction: [Activity] = ΔC = (ΔA 340 /min)/(ε app in mm) [Activity] = ΔC = (0.12/min)/(6.21 µmol/ml) = units/ml [Activity undiluted ] = (ΔC units/ml)(total Volume)(Dilution Factor) (Volume of enzyme used) [Activity undiluted ] = (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

10 Week 3: 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

11 Week 3: 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 ml 0.02 M KPO 4 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

12 Affinity Chromatography Week 3: Procedure Wash with KPO 4 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/KPO 4 Buffer Collect about fractions (1-2 ml each) Assay fractions for enzyme activity and protein content Pool up to 10 ml with highest activity What proteins come off in this step? Take small aliquot (~250 μl) of pooled fractions for activity and dye binding Why does LDH elute here?

13 Ultrafiltration Week 3: Procedure 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 Save aliquots of all fractions for Chapter 4 & 5!

14 Activity Assays Week 3: Procedure 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 ΔA 340 /min of What do you use to blank your spectrophotometer?

15 Protein Concentration Dye Binding Assay Find protein concentration for: 3P-Dialyzed Week 3: Procedure Pooled Affinity Fraction Pooled Concentrated Fraction Anything you did not finish in weeks 1 or 2 Use 1 ml aliquots for protein concentration A 595 should be within linear region of your standard curve Dilute protein when necessary What do you use to blank your spectrophotometer?

16 Lab Notebook Chapter 3 Purification details weights/volumes of each step Apparent extinction coefficient and spectra LDH activity assays A 340 vs. Time for 1 assay graph ΔA 340 /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

17 Purification Table Calculations Fraction Volume (ml) Corrected Volume (ml) Activity (units/ ml) Protein (mg/ml) Total Activity (units) Total Protein (mg) Specific Activity (units/ mg) Yield (%) ΔA340/ min Dilution Factor Homogenate S S P S P P-D Pooled Conc 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)

18 Final Example Purification Table Corrected Volume (ml) Activity (units/ml) Protein (mg/ml) Total Activity (units) Total Protein (mg) Specific Activity (units/mg) Yield (%) Fraction Homogenate 160 1S S P S P P-D Pooled Conc Any Questions on Chapter 3?