ANNOUNCEMENTS

• We LOVE to see that you are consulting with each other on the progress of your lab work and what everything means. COLLABORATION in this course is regarded very highly. HOWEVER, please make sure to respect the fine line between collaboration and non-individual work. Your TFs are starting to pick up “TOO SIMILAR” submissions.

I take ACADEMIC INTEGRITY VERY SERIOUSLY!!!

a. Please include your specific plans/ decisions about your own experimental set-up (esp. in terms of sample preI can give you feedback if I spot an issue

b. Your “discussion” with your partner is more efficient at the start of your lab time. • paration) in your pre-lab work so that:• Starting next week your pre-labs will be graded or re-assigned without comments

except for critical calculations or errors that will hinder your lab work.• If you submit before pre-lab but after the 9 am due time you will not be eligible for

revision.

Enzyme Purification Step

Volume(ml)

Activity (units/ml)

[protein] (mg/ml)

Total Activity (units)

Total Protein

(mg)

Specific Activity

(units/mg)

Yield %

Crude Extract (1S)

(2S)

(2P)

(3S)

(3P)

(3PD)

(3PD-FT)

(3PD-W)

(Emax)

(E pooled)

(E-concentrated)

MONITORING ENYME PURIFICATION

1.Cell Lysis (Homogenization)

2. Separation of soluble and

insoluble components of the

lysed cells (Centrifugation)

3. Separation of desired

protein from other proteins

(fractionation) by salting out

(Ammonium sulfate

precipitation)

4. Replacement/exchange

of protein environment

(dialysis)

5. Separation of desired protein

from other proteins (fractionation)

by column chromatography

(Affinity Chromatography)

LDH PURIFICATION FLOWCHART

Bovine Muscle or Heart Tissue

1P- precipitate(cell debris, nuclei)

1S- supernatant(crude extract- starting material)

Mince/ blend 4X 15 sec high pulseCentrifuge 15 min @14,500 rpm

Add saturated (NH4SO4 (aq) to 40% saturationcentrifuge 10 min @14,000 rpm

2P- precipitate 2S- supernatant

Redissolve in 15 ml KPO4

Dialyze

3P- precipitate 3S- supernatant

3PD- dialyzed

Perform Affinity Chromatography

W- wash E- eluateFT- flow through

Pool and concentrate

PURIFIED MUSCLE or HEART LDH

Corresponding general protein

purification step

WK3

What will you do?

● Run the dialyzed 3PD on an affinity chromatography column ● Determine which fractions have LDH activity● Pool them together and concentrate

MODULE 3C

Why is the procedure calling for a centrifugation step for your dialyzed sample before applying it to the column?

Cibacron blue F3GA dye

coupled to agarose

AFFI_GEL AFFINITY CHROMATOGRAPHYWhy does it work?

Binds to LDH at the

NADH binding site

Analog of AMP and binds to LDH at the

NADH binding site (Sulphates resemble

phosphate and ring resembles ribose)

Absorbs visible light appearing dark blue

NADH has a higher binding affinity to LDH than AMP

Dinucleoside diphosphate

NADH BINDING POCKET OF LDH

COLUMN PREPARATION

If column flow rate is very slow when you are draining the

equilibration buffer, do NOT add your sample, call your TF

Pipet 6 mlsof 50% slurry

Pipet 10-15 mls of

20mM KPO4

MODULE 3C- Affinity ChromatographyLoading

• 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

MAKE SURE TO KEEP ALL THE FRACTIONS ON ICE AT ALL TIMES

• 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 ChromatographyWashing

• 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 ChromatographyElution

• 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.

MODULE 3C- Affinity ChromatographyPooling Elution Fractions

CONCENTRATING POOLED ELUTIONS

15 mls 1-5 ml

• Obtain a pre-equilibrated concentrator (Vivaspin-15) 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.

THIS IS YOUR PRECIOUS PURE LDH

DATA TO FOCUS ON THIS WEEK

Enzyme Purification Step

Volume(ml)

Activity (units/ml)

[protein] (mg/ml)

Total Activity (units)

Total Protein

(mg)

Specific Activity

(units/mg)

Yield %

Crude Extract (1S)

(2S)

(2P)

(3S)

(3P)

(3PD)

(3PD-FT)

(3PD-W)

(Emax)

(E pooled)

(E-concentrated)