BY1101 Introduction to Molecular and Cellular Biology

BY1101 Introduction to Molecular and Cellular Biology

Tutorial for module BY1101:

Chromatography

Joe Colgan ([email protected])

mailto:[email protected]

Tutorial objectives

• Describe chromatography • Describe the different types and why they

were used in the BY1101 practicals


Column chromatography• What is chromatography?• A set of lab techniques to separate mixtures

• Mobile phase: • Fluid that houses the mixture to be separated• (e.g. Cell lysate, haemoglobin, mould extract)

• Stationary phase: • Structure holding another material that interacts with

and aids in separation of mixture• (e.g. Sepharose, sephadex, DEAE-cellulose)


Mobile

Stationary

Column chromatography


Affinity chromatography

Gel filtrationchromatography

Ion exchangechromatography

Separates molecules based on biological

specificity

Separate molecules based on size

Separates molecules based on charge

Practical 2 Practical 3Experiment 1

Practical 3Experiment 2

Three types of column chromatography used in the BY1101 practicals. What are they?

Affinity chromatographyBY1101 Introduction to Molecular and Cellular Biology

Purpose•Purification and concentration of biomolecules, such as proteins, on the basis of their biological specificity

Applications•Purification of antibodies•Purification of enzymes

What do we use affinity chromatography for?


Glutathione S-transferase (GST): • Catalyzes conjugations of the substrate glutathione (GSH)

•GST is an enzyme and binds GSH in an enzyme-substrate complex

Enzyme (E) + Substrate (S) Enzyme-substrate complex

•Interaction is dynamic and GST will bind reversibly

What was the purpose of Practical 2 – Experiment One?

EnzymeSubstrate Enzyme-substrate complex

GSH GST


What is the source of the GST?

Natural source Recombinant protein


l P O gst gene

mRNA

Transcription

Translationl= lac operonP= PromoterO= Operator

Affinity chromatographyWhat is a recombinant protein?A protein encoded for by a gene – recombinant DNA – that has been cloned into a system that supports its transcription and translation

Repressor protein

l P O gst gene

mRNA

l= lac operonP= PromoterO= Operator



Transcription of mRNA

IPTG

How do we get “overexpression” of a protein?Isopropyl-beta-D-thio-galactoside (IPTG):IPTG binds to the repressor protein and inactivates it

Repressor protein

Within the present experiment, what would be the purpose of adding IPTG to the bacterial culture?


Molecule of interest Mobile phase Stationary phase

Escherichia coli(bacterial) lysate

Sepharose beads coated with glutathione

Glutathione S-transferase

Are the proteins present in the cell lysate in their native (active) state or are they denatured?

Would you expect the proteins present in the lysate to exhibit their natural biological activity?

Why was it important to keep the cell lysate on ice?


Affinity chromatographyCell lysate Glutathione S-

transferase (GST)

Sepharose Glutathione (GSH)

Sepharose-Glutathione

Stationary phase Mobile phase


1. Pour the column(Sepharose Beads-Stationary phase)

2. Wash the column (Phosphate buffered saline)

Stationary

Mobile


1 2 3 4 5 6

3. Run the column(E. coli lysate) Contains enzyme of interest- GST within lysate binds to glutathione-sepharose beads complex

4.(PBS) Washes the column- Other bacterial proteins are washed out of the column leaving only GST bound to sepharose beads

Mobile

Stationary

Why does GST remain in the column after the PBS washes?

Would you expect GST to be in fraction one?


1 2 3 4 5 6 7

3. Run the column(E. coli lysate) Contains enzyme of interest- GST within lysate binds to glutathione-sepharose beads complex

4. (PBS) Washes the column- Other bacterial proteins are washed out of the column leaving only GST bound to sepharose beads

5. (Glutathione) Substrate of enzyme- High concentration of glutathione displaces GST from the beads, binds to GST and is eluted out of the column

Mobile

Stationary


1 7All proteins in lysate except GST

PurifiedGST

GST binds to GSH-Sepharose beads

Free GSH bind GST and elutes

Addition of free GSH

Column is washed

Non-bound proteins removed


SDS-PAGE preparation• Precipitation of protein with trichloroacetic acid (TCA)

• Pellet the precipitated protein by centrifugation

• Dissolve precipitated protein in sodium-dodecyl sulphate (SDS)

• Boil the protein samples


Gel filtration chromatographyBY1101 Introduction to Molecular and Cellular Biology

PurposeSeparation of macromolecules based on size

Applications•Determination of relative molecular size•Separation of molecules on the basis of size•Removal of inorganic ions from preparation of protein

Practical 3- Experiment One


Gel filtration chromatography

Direction of flow


Gel filtration chromatographyPractical 3- Molecule of interest

Haemoglobin Oxyhaemoglobin

Purple/red colourOxygen-depleted blood

Venous blood

Scarlet/red colourOxygen-rich blood

Arterial blood


Practical 3- Molecule of interest

Gel filtration chromatography

Haemoglobin Methaemoglobin Haemoglobin

Oxidation Reduction

Oxidation: Potassium ferricyanide + Haemoglobin Oxidised haemoglobin (methaemoglobin)

Reduction: Ferrous sulphate + methaemoglobin Reduced haemoglobin


1. Pour the column(G-25 Sephadex beads-Stationary phase)

2. Wash the column (20mM PBS, pH 7)

Stationary

Mobile

3. Add the reducing agent(40mM FESO4 + 80mM Na2EDTA)

4. Add methaemoglobin

Methaemoglobin

Reducing agent

Haemoglobin


Stationary

Mobile


Gel filtration chromatographyAddition of reducing

agentAddition of

methaemoglobinReduction of

methaemoglobin to haemoglobin

Ion exchange chromatographyBY1101 Introduction to Molecular and Cellular Biology

PurposeSeparation of molecules on the basis of charge

Applications•Water softening, purification and decontamination

Ion exchange chromatographySeparation of molecules on the basis of charge

Dire

ction

of fl

ow



Glucose oxidase

Catalase

Molecules of interest Mobile phase Stationary phase

Aspergillus niger (fungal) extract

DEAE- Cellulose

• Stationary phase: • Diethylaminoethyl (DEAE) cellulose


Ion exchange chromatography

• Positively charged (protonated)• Interacts with negatively charged molecules (anions)• Anion exchanger: Stationary phase is positively charged


Glucose oxidase

Separation of molecules on the basis of charge

Ion exchange chromatography

Glucose oxidase: •Oxidation of glucose to hydrogen peroxide and glucono-1,5-lactone, which hydrolyzes to gluconic acid •Glucose oxidase (GO) requires cofactor flavin adenine dinucleotide (FAD)

Glucose + GO:FAD Glucono-1,5-lactone + GO:FADH2

GO:FADH2 + O2 GO:FAD + H2O2


Glucose Glucose oxidase

Catalase


Catalase: •Catalyzes the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2)

Separation of molecules on the basis of charge

2H2O2 2H2O + O2


Hydrogen peroxide Catalase


1. Pour the column(DEAE-Cellulose-Stationary phase)

2. Wash the column(PBS)

Mobile

Stationary

3. Run the column(Mould extract-Aspergillus niger)

4. Wash the column(Buffer 1- 20mM NaOAc, 5mM acetic acid)

Mobile

Stationary


How do we get our enzymes of interest out of the column?

1 2 3 4 5 6 7

3. Run the column(Mould extract-Aspergillus niger)

4. Release the bound molecules(Buffer 1- 20mM NaOAc, 5mM acetic acid)(Buffer 2- 40mM NaOAc, 40mM acetic acid)(Buffer 3-100mM NaOAc, 100mM acetic acid)

Mobile

Stationary


• Lowering pH neutralizes negative charge on the protein molecules

• Increase anionic molecules for competition


Buffer II Buffer III

Glucose oxidaseCatalase

Buffer II elutes catalase

40mM sodium acetate

40mM acetic acid

Buffer III elutes glucose oxidase

100mM sodium acetate

100mM acetic acid

Buffer I

20mM sodium acetate

5mM acetic acid


Ion exchange chromatographyAddition of different

buffers changes chargeNegatively charged

molecules bind to beadsAdd molecules of

varying ionic charge


Glucose GlucoseHydrogen peroxide

(H2O2)

Hydrogen peroxide

(H2O2)

Catalase Glucose oxidase

ControlControl

Functional assaysIn biological studies, what is the role of control samples?


SummaryChromatography: Used to separate out

mixturesAffinity

chromatographyGel filtration

chromatographyIon exchange

chromatography

Separates molecules based on biological

specificity

Separate molecules based on size

Separates molecules based on charge

MCQ Advice

• Get your lab books up to date (e.g. Tables, graphs)

• If you have problems with the questions ask a demonstrator (or me)

• When it comes to the exam, revise all of the lab book (including the introductory notes)

• Read over lab slides available on the teaching website


Next week

- Developmental biology: Embryology- Lectures 3, 4 and 5- Campbell: Chapter 47 (section 47.1)


BY1101 Introduction to Molecular and Cellular Biology

Documents

cellular biology tutorial

operatorby1101 introduction

substrate glutathione

module by1101

cell lysate

present experiment

types of column chromatography

purpose of practical