ANNOUNCEMENTS• Please use the following resources for your post lab work:

a. Backgroud section of each Laboratory Module in your ELNb. Excel tutorials posted under the “Reference Materials” tab in your ELNc. “Guide to preparing professional exhibits” document posted under the

“Reference Materials” tab in your ELN.

• Please consider making use of the weekly “Skill Enhancement Laboratory hours”. Sign up 24 hours before: https://docs.google.com/document/d/1tSZQiOtFGtCf8OKzD5FIvyPF1Mpo8pG461-5H0aQe-M/edit?usp=sharing

• Please make sure that all your lab work for this course is in a single physical notebook since you will be expected to look back on previous weeks’ work routinely.

• This week is the start of your 7 week protein purification and characterization module. Please ARRIVE ON TIME, COME PREPARED and STAY ON TOP OF YOUR LAB WORK WEEKLY. TAKE THE TIME TO PLAN AND STRATEGIZE WITH YOUR TEAM MATE(S) BEFORE YOU START YOUR EXPERIMENTS.

RECOMMENDATIONS FOR CH3 LAB WORK

• This week is the start of your multi-week LDH purification that will be followed by its analysis. Each week will be a TEAM effort and effective teamwork/ collaboration is one of the skills we expect you to develop and is a part of your lab assessment (directly and indirectly). Please ARRIVE ON TIME, COME PREPARED and TAKE THE TIME TO PLAN AND STRATEGIZE WITH YOUR TEAM MATE(S) BEFORE YOU START YOUR EXPERIMENTS

• Stay on top of your lab work WEEKLY even when there is no report to be turned in. It is your responsibility as a team to identify issues from the previous week, foresee the potential consequences, and seek proactive help during OHs (not emails).

Enzyme Purification

Isolation of Lactate Dehydrogenase from Bovine Tissue as an example

https://my.bpcc.edu/content/blgy225/EnergyInTheHumanBody/lactic%20acid%202.jpg

Last step of

anaerobic glycolysis

Found as several

isozymes

+

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

SUCCESS CRITERIA FOR ENYME PURIFICATION

1. HIGH PROTEIN YIELD (total amount of purified protein)

What to do: BE PREPARED, BE CAREFUL, THINK BEFORE YOU ACT, BE

ORGANIZED and DON”T THROW OUT CRITICAL FRACTIONS

(keep everything in the box given to you and keep a good

record in your notebook)

2. HIGH SPECIFIC ACTIVITY

(activity of desired enzyme/ total amount of protein)

What to do: KEEP ENZYME COLD, AVOID BUBBLES

In any successful enzyme purification, each consecutive

step should increase specific activity

3. LOW COST (time & money)

What to do: MINIMIZE # OF STEPS, SELECT THE LEAST EXPENSIVE METHOD

MODULE 3A

1.Prepare Crude Extract from either bovine heart or skeletal muscle

(Your TFs will assign you for muscle or heart)

2. Obtain spectra of NAD+ and NADH to determine app of NADH

3. Assay LDH activity of crude extract

You’ll keep using this assay !!!

Make sure to master it this week.

PART 1: Preparation of Crude Extract

1. Blending (Homogenization) & Filtering:

Why does it work? Cell membrane is fragile and is disrupted by

physical stress releasing inner contents into the experimental

medium. Filtering eliminates big insoluble particles

+ K/PO4 buffer +

2. Centrifugation:

Why does it work? Centrifugal force to

separates soluble components (supernatant)

from insoluble cell debris (pellet) due to

density differences

STEPS FOR WEEK1

1S COLLECT

1P DISCARD

Tips and Advice for Preparing Crude Extract

• Before centrifugation, balance your two tubes and record the total volume that went into the tubes.

• Do NOT overfill tubes (max 45 mls). Put any homogenate volume that exceeds 90 mls into a separate tube (record volume) and do NOT process further.

• Record volume information and your observations at each step of the procedure

• Blend your tissue pieces well- no chunks and keep cold during the process.

• Collect cude extract (1S) into 50 ml conical tubes LABELED with the Sample Info, you and your partner’s names and date. Make sure to leave room for expansion during freezing. Keep it on ice and then freeze at -20C.

• Take three 250 ul aliquots of 1S into two LABELED 1.5 ml tubes to use for activity and dye binding assays. Freeze two immediately in your box at -20C and keep the other on ice until the end of the lab period. Mark the cap as used (*) and then freeze it in the same box as the first two.

PART 2: Absorption spectra of NAD+ and NADH

Underlying Principle

WHY do you need Part 2?

Abs C through AS LONG AS L is constant

A=(ε)(l)(c)Obtain UV-vis

spectra (250-400

nm for NAD+

and NADH

using micro-UV

plastic cuvettes

Measure absobance

of NAD+ and NADH

at 340 nm on

benchtop specs using

glass test tubes

PART 3: ASSAY FOR LDH ACTIVITY

Make sure to include in your notebook operating

instructions for performing kinetic assays in the Genesis

10 spectrophotometer as well as how to display rate data

In the LDH Activity Assay you will be measuring the change in absorbance at 340 nm (A340) over a predetermined amount of time.You will use this information to calculate the rate of decrease in absorbance (A340/min) which is the measure for the rate of consumption of reactant NADH (rate of reaction)

LDH ACTIVITY ASSAY

LDH ACTIVITY ASSAY

using enzyme dilution buffer

LDH ACTIVITY ASSAY TIPS

• DO NOT put COLD cocktail in the spec! Condensation will interfere with imstrument readings. You can keep the cocktail at RT for an hour before NADH starts oxidizing.

• Determining the “optimal” enzyme dilution for the assay is a process of trial and error. So this week you are asked to try several dilutions to get a feel for it.

• Use “Enzyme Dilution Buffer (EDB)” to dilute the enzyme.• Blank he spec with EDB (Why not cocktail?)• Work FAST. The reaction starts as soon as you put the enzyme in the tube.

So get your tube in the spec and start run ASAP to obtain most accurate rate.• Ask TFs how to adjust plot on spec to get slope from a selected linear region.

PART 4: Bradford Assay for total protein mass

To obtain accurate protein mass determination using Bradford assay you need to be collecting data to determine a new standard curve EVERY time you run a new sample. But if you only want a general estimate you can use a previous standard curve you made.

Depending on how much time you have either:1. Set up 6 tubes of BSA standards (which ones do you think they shoud be?) and

all four of your crude extract dilutions using 25 ul of each sample.2. Only set up two test tubes corresponding to the 1:400 and 1:800 dilutions of

your crude extract using 25 ul of the samples.

LDH ACTIVITY CALCULATIONS

LDH ACTIVITY CALCULATIONS

TIPS FOR SUCCESS

Make sure to use your In-Lab work time efficiently

each week to engage in ongoing discussions with

your partner, other groups and your TFs about how

your purification is progressing.

MODULE 3A CHECKLIST

At the end of the lab you should have:

• Record of the ID of your starting material

• Record of volume and observational information for your material at

each step of your purification.

• UV-Vis data for NAD+ and NADH and A340 record for NADH on

benchtop spect. to calculate apparent extinction coefficient

• Enzyme Activity data for 1S crude extract fraction

- Recorded rates for the following dilutions: undiluted, 1:20, 1:200,

1:400, 1:800 with A340 =-0.05-0.25 range

- Record of tabular data (time vs A340) for 1:400 dilution.

• Bradford data for your crude extract (1S) sample

NOTE: You will only be measuring the Enzyme activity of the crude

extract this week.

• Labeled and frozen 1S crude extract fractions (three small aliquots and

two 50 ml tubes)