SSodium odium DDodecylodecylSSuulphlphatat

e-e- PPolyolyAAcrylamide crylamide

GGel el EElectrophoresislectrophoresis

((SDS-PAGESDS-PAGE))Exercise Exercise 1010

Experimental GoalsExperimental Goals

To understand the principle of SDS-To understand the principle of SDS-PAGEPAGE

To become familiar with the SDS-To become familiar with the SDS-PAGE setupPAGE setup

Electrophoresis is a laboratory technique for

separating molecules based on their charge

What is Electrophoresis?

Charged molecules are separated based on their electrical charge and size within a matrix

Separation of a Mixture of Charged Molecules

Charge Separation

Size Separation

Analyze

Identify

PurifyMixture of Charged Molecules

Positive Molecules

Negative Molecules

The gel (matrix)The gel (matrix)

The gel (matrix) itself is composed of The gel (matrix) itself is composed of either agarose or polyacrylamide. either agarose or polyacrylamide.

Polyacrylamide is a cross-linked Polyacrylamide is a cross-linked polymer of acrylamide. polymer of acrylamide. Acrylamide is a potent neurotoxin and Acrylamide is a potent neurotoxin and

should be handled with care! should be handled with care!

Polyacrylamide gelsPolyacrylamide gels

Have smaller pores than agarose, Have smaller pores than agarose, therefore high degree of resolving therefore high degree of resolving power. power.

Can separate DNA fragments which Can separate DNA fragments which range in size from 10-500 bp.range in size from 10-500 bp.

DNA fragments which differ in size by DNA fragments which differ in size by one nucleotide can be separated from one nucleotide can be separated from each other.each other.

Polyacrylamide gel electrophoresis is Polyacrylamide gel electrophoresis is also used to separate protein molecules.also used to separate protein molecules.

Protein Protein ElectrophoresisElectrophoresis

Separate proteins based on Separate proteins based on Size (Molecular Weight - MW)Size (Molecular Weight - MW)

Allows us toAllows us to characterize characterize quantifyquantify determine purity of sampledetermine purity of sample compare proteins from different compare proteins from different

sourcessources And it is a step in Western blotAnd it is a step in Western blot

Protein Protein ElectrophoresisElectrophoresis

Proteins, unlike DNA, do not have a Proteins, unlike DNA, do not have a constant size to charge ratioconstant size to charge ratio In an electric field, some will move to the In an electric field, some will move to the

positive and some to the negative pole, positive and some to the negative pole, and some will not move because they are and some will not move because they are neutralneutral

Native proteins may be put into gel Native proteins may be put into gel systems and electrophoresedsystems and electrophoresed

An alternative to native protein gels forces An alternative to native protein gels forces all proteins to acquire the same size to all proteins to acquire the same size to charge ratiocharge ratio

SDS-PAGESDS-PAGE

SDS-PAGESDS-PAGE (( sodium dodecylsusodium dodecylsulphlphate-ate-ppolyacrylamide gel electrophoresis)olyacrylamide gel electrophoresis)

The purpose of this method is to separate The purpose of this method is to separate proteins according to their size, and no proteins according to their size, and no other physical featureother physical feature

In order to understand how this works, we In order to understand how this works, we have to understand the two halves of the have to understand the two halves of the name: name: SDSSDS and and PAGEPAGE

SSodium odium DDodecylsuodecylsulphlphateate Since we are trying to separate many Since we are trying to separate many

different protein molecules of a different protein molecules of a variety of shapes and sizes, variety of shapes and sizes, we first want to get them to be linear we first want to get them to be linear no longer have any secondary, tertiary or no longer have any secondary, tertiary or

quaternary structure (i.e. we want them quaternary structure (i.e. we want them to have the same linear shape). to have the same linear shape).

Not only the mass but also the shape Not only the mass but also the shape of an object will determine how well it of an object will determine how well it can move through and environment. can move through and environment.

So we need a way to convert all So we need a way to convert all proteins to the same shape - we use proteins to the same shape - we use SDS.SDS.

SSodium odium DDodecylsuodecylsulphlphateate

SDS (sodium dodecyl sulfate) is a SDS (sodium dodecyl sulfate) is a detergent that can dissolve detergent that can dissolve hydrophobic molecules but also has hydrophobic molecules but also has a negative charge (sulfate) attached a negative charge (sulfate) attached to it. to it.

If SDS is added to proteins, they If SDS is added to proteins, they will be soluablized by the detergent, will be soluablized by the detergent, plus all the proteins will be covered plus all the proteins will be covered with many negative charges.with many negative charges.

SSodium odium DDodecylsuodecylsulphlphateate

A sample of protein, often freshly isolated and unpurified, A sample of protein, often freshly isolated and unpurified, is boiled in the detergent sodium dodecyl sulfate and beta-is boiled in the detergent sodium dodecyl sulfate and beta-mercaptoethanolmercaptoethanol The mercaptoethanol reduces disulfide bondsThe mercaptoethanol reduces disulfide bonds The detergent disrupts secondary and tertiary structureThe detergent disrupts secondary and tertiary structure

The end result has two important features: The end result has two important features: 1.1. all proteins contain only primary structure and all proteins contain only primary structure and 2.2. all proteins have a large negative charge which means they all proteins have a large negative charge which means they

will all migrate towards the positive pole when placed in an will all migrate towards the positive pole when placed in an electric field.electric field.

They migrate through a gel towards the positive pole at a They migrate through a gel towards the positive pole at a rate proportional to their linear sizerate proportional to their linear size Molecular weights with respect to size markers may then be Molecular weights with respect to size markers may then be

determineddetermined

SSodium odium DDodecylsuodecylsulphlphateate

Now we are ready to focus on the second half - PAGE.

SDS and ProteinsSDS and Proteins

SDS

Protein

SDS and ProteinsSDS and Proteins

So much SDS binds to proteins that the So much SDS binds to proteins that the negative charge on the SDS drowns out any negative charge on the SDS drowns out any net charge on protein side chainsnet charge on protein side chains

In the presence of SDS all proteins have In the presence of SDS all proteins have uniform shape and charge per unit lengthuniform shape and charge per unit length

SDS nonpolar chains arrange themselves on SDS nonpolar chains arrange themselves on proteins and destroy secondary tertiary and proteins and destroy secondary tertiary and quarternary structrurequarternary structrure

Polyacrylamide Gel Polyacrylamide Gel Electrophoresis (PAGE)Electrophoresis (PAGE)

PAGE is the preferred method for PAGE is the preferred method for separation of proteinsseparation of proteins

Gel prepared immediately before use Gel prepared immediately before use by polymerization of acrylamide and by polymerization of acrylamide and N,N'-methylene bis acrylamide.N,N'-methylene bis acrylamide.

Porosity controlled by proportions of Porosity controlled by proportions of the two components. the two components.

Catalyst of Catalyst of polymerizationpolymerization

Polymerization of acrylamide is Polymerization of acrylamide is initiated by the addition of initiated by the addition of ammonium persulphate and the base ammonium persulphate and the base N,N,N’,N’-N,N,N’,N’-tetrametyhlenediamine tetrametyhlenediamine (TEMED)(TEMED)

TEMED catalyses the decomposition TEMED catalyses the decomposition of the persulphate ion to give a free of the persulphate ion to give a free radicalradical

Polymerization of Polymerization of acrylamideacrylamide

Polymerization of Polymerization of acrylamideacrylamide

Temed

Cross-linked Cross-linked polyacrylamide gels polyacrylamide gels are formed from the are formed from the polymerisation of polymerisation of acrylamide monomer acrylamide monomer in the presence of in the presence of smaller amounts of smaller amounts of N,N’-N,N’-methylenebisacrylamimethylenebisacrylamide (bis-acrylamide)de (bis-acrylamide)

Bisacrylamide is the Bisacrylamide is the most frequently used most frequently used cross linking agent for cross linking agent for polyacrylamide gelspolyacrylamide gels

Polyacrylamide GelsPolyacrylamide Gels Bis-Acrylamide polymerizes along with acrylamide Bis-Acrylamide polymerizes along with acrylamide

forming cross-links between acrylamide chainsforming cross-links between acrylamide chains

Polyacrylamide GelsPolyacrylamide Gels Pore size in gels can be varied by varying Pore size in gels can be varied by varying

the ratio of acrylamide to bis-acrylamidethe ratio of acrylamide to bis-acrylamide

Lots of bis-acrylamideLittle bis-acrylamide

Protein separations typically use a 29:1 Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratioor 37.5:1 acrylamide to bis ratio

Side view Side view

Movement of Proteins in Movement of Proteins in GelGel

Movement of Proteins in Movement of Proteins in GelGel

smaller proteins smaller proteins will move will move through the gel through the gel faster while faster while larger proteins larger proteins move at a move at a slower paceslower pace

Components of the SystemComponents of the System

DC Power Source, Reservoir/Tank, DC Power Source, Reservoir/Tank, Glass Plates, Spacers, and Combs Glass Plates, Spacers, and Combs

Support mediumSupport medium Gel (Polyacrylamide)Gel (Polyacrylamide)

Buffer SystemBuffer System High Buffer CapacityHigh Buffer Capacity

Molecules to be separatedMolecules to be separated ProteinsProteins Nucleic AcidsNucleic Acids

Reservoir/TankPower Supply

Glass Plates, Spacers, and Combs

Vertical Gel Format: Polyacrylamide Vertical Gel Format: Polyacrylamide Gel ElectrophoresisGel Electrophoresis

Step by Step Instructions on how to assemble the polyacrylamide gel apparatus

ProcedureProcedure

Prepare polyacrylamide gelsPrepare polyacrylamide gels Add Add diluted samplesdiluted samples to to the samplethe sample bbufferuffer Heat to 95Heat to 95C for C for 44 minutes minutes Load Load the samples onto polyacrylamide the samples onto polyacrylamide

gelgel Run at 200 volts for 3Run at 200 volts for 30-400-40 minutes minutes Stain Stain

Gel PreparationGel Preparation

ReagentReagent 8% (Running 8% (Running Gel)Gel)

5% (Stacking 5% (Stacking Gel) Gel)

Acrylamide/ Acrylamide/ Bisacrylamide Bisacrylamide

(40%) * (40%) *

4.0 mls4.0 mls 2.5 mls2.5 mls

1 M Tris-HCl pH 8.81 M Tris-HCl pH 8.8 7.5 mls7.5 mls 7.5 mls7.5 mls

water (distilled)water (distilled) 8.2 mls8.2 mls 9.7 mls9.7 mls

10% SDS10% SDS 200 200 µµll 200 200 µµll

10% Ammonium 10% Ammonium PersulfatePersulfate

100 100 µµll 100 100 µµll

TEMED (added last)TEMED (added last) 10 10 µµll 10 10 µµll

* = 19:1 w:w ratio of acrylamide to N,N'-methylene bis-* = 19:1 w:w ratio of acrylamide to N,N'-methylene bis-acrylamide acrylamide

Gel PreparationGel Preparation

Mix ingredients Mix ingredients GENTLY!GENTLY! in the order in the order shown above, ensuring no air bubbles shown above, ensuring no air bubbles form. form.

Pour into glass plate assembly Pour into glass plate assembly CAREFULLY. CAREFULLY.

Overlay gel with isopropanolOverlay gel with isopropanol to to ensure a flat surface and to exclude air. ensure a flat surface and to exclude air.

Wash off isopropanol with water after Wash off isopropanol with water after gel has set (gel has set (++15 min). 15 min).

Sample BufferSample Buffer

TrisTris buffer to provide appropriate pH buffer to provide appropriate pH SDS SDS (sodium dodecyl sulphate)(sodium dodecyl sulphate)

detergent to dissolve proteins and give detergent to dissolve proteins and give them a negative chargethem a negative charge

GlycerolGlycerol to make samples sink into wells to make samples sink into wells Bromophenol BlueBromophenol Blue dye to visualize dye to visualize

samplessamples

Heat to 95Heat to 95C for C for 44 minutes minutes

Loading Samples & Running the Loading Samples & Running the gelgel

Run at 200 volts for 3Run at 200 volts for 30-400-40 minutes minutes

Running Buffer, pH 8.3Running Buffer, pH 8.3 Tris Base       12.0 g Tris Base       12.0 g Glycine          57.6 gGlycine          57.6 gSDS                4.0 gSDS                4.0 g

distilled water to 4 literdistilled water to 4 liter

SDS-PAGESDS-PAGE

Staining Proteins in GelsStaining Proteins in Gels

Chemical stains detect proteins based on Chemical stains detect proteins based on differential binding of the stain by the differential binding of the stain by the protein molecules and the gel matrix. protein molecules and the gel matrix.

They are nonspecific in action, detecting They are nonspecific in action, detecting proteins without regard to their individual proteins without regard to their individual identities. identities.

The important characteristics for a useful The important characteristics for a useful stain are: low background, high stain are: low background, high sensitivity, large linear range and ease of sensitivity, large linear range and ease of use.use.

Staining Proteins in GelsStaining Proteins in Gels

How to Quantify Proteins ?• Densitometry

• Coomassie Brilliant Blue •The CBB staining can detect about 1 µg of protein in a normal band.

• Silver Staining •The silver stain system are about 100 times more sensitive, detecting about 10 ng of the protein.

Molecular Weight Standard

250 KD150100755037

25

20

15

10

Molecular weight estimation by SDS-Molecular weight estimation by SDS-PAGEPAGE

Molecular weight estimation by SDS-Molecular weight estimation by SDS-PAGEPAGE

Calibration curve for molecular weight estimation.

Western BlottingWestern Blotting