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)

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

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

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

Drain Buffer to surface,

close stopcock

Pipet in all resin on side of

column, open stopcock, let

settle, but DO NOT let column

dry out!

Equilibrate column with

buffer

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

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

3P–Dialyzed

Affinity Purification

Wash Fractions – Non-specifically bound

proteins

NADH Eluted Fractions – Specifically bound to

resin

Ultrafiltration – removes buffer

Purified LDH

Flow Chart for LDH Purification

See flow chart p. 70

Activity Assay for total number

of units!

Load 5,000 Units on to

column Wash fractions with KPO4

Buffer Elute fractions

with KPO4/NADH Buffer

Pool fractions with highest

activity

Concentrate samples to ~1 ml

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!

Week 3: 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

● 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

● Load 10.8 ml on to affinity column

● If you have > 5000 units, load all but 250 μl aliquot

Week 3: Procedure

6957 units

● 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

Week 3: Procedure

Week 3: 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

– 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

What proteins come off in this

step?

Why does LDH elute here?

Week 3: 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

Save aliquots of all fractions for Chapter 4 & 5!

● 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

– 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?

Week 3: Procedure

ALL POOLED FRACTIONS

● 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?

Week 3: Procedure

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

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 160 160

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)

Fraction

Corrected Volume

(mL) Activity

(units/mL) Protein

(mg/mL)

Total Activity (units)

Total Protein

(mg)

Specific Activity

(units/mg) Yield (%)

Homogenate 160

1S 160.0 272.5 125 43594 20000 2.2 100.0

2S 60.0 456.0 30 27362 1800 15.2 62.8

2P 30.0 10.0 44 301 1320 0.2 0.7

3S 70.0 12.0 17 839 1190 0.7 1.9

3P 34.0 560.4 26 19053 884 21.6 43.7

3P-D 36.0 510.1 22 18365 792 23.2 42.1

Pooled 32.0 499.7 4 15991 128 124.9 36.7

Conc. 8.0 1959.4 15 15675 120 130.6 36.0

Final Example Purification Table

Any Questions on Chapter 3?