dr. a. khaleel ahamed associate professor of botany jamal mohamed college

Dr. A. Khaleel AhamedAssociate Professor of Botany

Jamal Mohamed CollegeTiruchirappalli – 620 020

khaleeljmc@gmail.comMobile No. +919443065561

Gel Electrophoresis

• An essential method to separate a mixture of charged molecules (DNA, proteins)

• Principle: migration in electric field and retardation by surrounding matrix

• Common matrices for protein gel electrophoresis:

AgarosePolyacrylamide

1D-Electrophoresis (SDS-PAGE)

• Denaturation and reduction

• Single amino acid chains

• Separation by molecular mass

• All proteins migrate towards anode

Applications of 2-D electrophoresis

• Analysis of cell differentiation• Detection of disease markers• Cancer research• Purity checks and • Microscale protein purification.

Experimental sequences for 2D electrophoresis

1. Sample preparation2. IPG strip rehydration3. IEF4. IPG strip equilibration5. SDS-PAGE6. Visualization and 7. Analysis

Advantages of 2-D Electrophoresis

• Tolerant to crude sample loads:no pre-purification (like chromatography)has to be employed.• ?? Highly resolution.• ?? Very effective fraction collectors• ?? Proteins are protected inside the gelmatrix

Sample Preparation

• Cell washing• ?? Cell disruption• ?? Protein precipitation• ?? Solubilization• ?? Protection against protease activities• ?? Removal of

- nucleic acids- lipids- salts, buffers, ionic small molecules- insoluble material

Cell washing

• To remove contaminant material.• Frequent used buffer- PBS (phosphate buffer saline): sodium chloride, 145 mM (0.85%) in phosphate buffer, 150 mM, pH7.2- Tris buffer sucrose (10mM Tris, 250 mM sucrose, pH 7,2)• Enough osmoticum to avoid cell lysis

Cell disruption methods

Gentle lysis method1. Osmotic lysis (cultured cells)?? Suspend cells in hypoosmotic solution.2. Repeated freezing and thawing (bacteria)Freeze using liquid nitrogen3. Detergent lysis (yeast and fungi)Lysis buffer (containing urea and detergent)SDS (have to be removed before IEF)4. Enzymatic lysis (plant, bacteria, fungi)Lysomzyme (bacteria)Cellulose and pectinase (plant)Lyticase (yeast)

Cell disruption (continued)

Vigorous lysis method1. Sonication probe (cell suspension)Avoid overheat, cool on ice between burst.2. French pressure (microorganism with cell wall)Cells are lysed by shear force.3. Mortar and pestle (solid tissue, microorganism)Grind solid tissue to fine powder with liquid nitrogen.4. Sample grinding kit (for small amount of sample)For precious sample.5. Glass bead (cell suspension, microorganism)Using abrasive vortexed bead to break cell walls.

Cell disruption (continued)

Key variables for successful extraction fromcrude material1. The method of cell lysis2. The control of pH3. The control of temperature4. Avoidance of proteolytic degradation

Removal of contaminants

Major type of contaminants:1. DNA/RNA2. Lipids3. Polysaccharides4. Solid material5. Salt

DNA/RNA contaminant

• DNA/RNA can be stained by silver staining.• They cause horizontal streaking at the acidic part of

the gel.• They precipitate with the proteins when sample

applying at basic end of IEF gel• How to remove:

1. Precipitation of proteins2. DNase/RNase treatment3. Sonication (mechanical breakage)4. DNA/RNA extraction method (phenol/chroloform)

Removal of other contaminants

• Removal of lipids:>2% detergentPrecipitation

• Removal of polysaccharides:

Precipitation

• Removal of solidmaterialCentrifugation

• Removal of saltsMicrodialysisPrecipitation

Protein precipitation

Ammonium sulfate precipitation:De-salting necessaryTCA precipitation:Can be hard to resolubilizeAcetone and/or ethanol:many proteins not precipitatedTCA plus acetone:More effective than either alone, good for basicproteins

Protein solubilization

• Urea (8-9.8 M), or 7 M urea / 2 M thiourea• Detergent (CHAPS,…)• Reductant (DTT, 2-mercaptoethanol)• Carrier ampholytes (0.8 % IPG buffer)• Sonication can help solubilization

ReductantsDTT (dithiothreitol)

most commonly used

DTE (dithioerythreitol)

interchangeable with DTT

2-mercaptoethanol required at high concentration, contains impurities

Tributylphosphine Poorly soluble, very hazardous

Triscarboxyethylphosphine

Good reductant, but negative charge makesit unsuitable for 1st dimension.

Triscyanoethylphosphine

Uncharged, soluble, but efficacy as reductant is in doubt.

Protease inhibitorsPMSF(phenylmethyl sulfonylfluoride)

Most commonly usedInactivates serine and cysteine proteases

AEBSF (Pefabloc) More soluble, less toxic than PMSF, but can cause charge modifications(?).

EDTA Inhibits metalloproteases

High pH Inhibits most proteases, but avoid Trisbase

De-salting techniques

• Dialysis• Gel filtration• Precipitation/

resuspension

• Slow• Protein losses• Complicated, can cause

losses

FIRST DIMENSION

First Dimension: Denaturing IEF

• High molar (8 mol/L) urea, thiourea- one conformation of a protein- for protein solubility- prevents protein aggregates and hydrophobic interactions• Non-ionic or zwitterionic detergent

- for protein solubility• IPG Buffer (carrier ampholyte mixture)

- for protein solubility- raises the conductivity of the DryStrips• DTT, DTE (no 2-mercaptoethanol)

- prevents different oxidation steps

IEF with Carrier Ampholytes

Plot of the net charge of a protein versus the pH of its environment

Immobiline DryStrips: 1st Generation

• 11 cm strips:pH 4 - 7pH 3 - 10pH 6 - 11• 7 cm, 13 cm and 18 cm strips:

pH 4 - 7pH 3 - 10 L (linear gradient)pH 3 - 10 NL (non-linear gradient)pH 6 - 11

Wide and Narrow pH Gradients

• Wide gradients are applied for entire protein spectrum

• Narrow gradients are applied for:

- increased resolution- increased loading capacity

Guidelines for choosing ImmobilineDryStrip gels

Ettan IPGphor

The IPGphor Platform

IPGphor Strip Holder

IPGphor features

• Platform accommodates up to 12 strip holders• 7, 11 , 13 , 18 and 24 cm strip holders• Cup-loading stripholders for all lengths• Built-in power supply delivering 8000 V, 1.5 mA• Built-in Peltier cooling, 18 - 25 °C• Programmable “delayed start” rehydration period• 10 possible programs, 10 phases each (ramp or step)• Safety lid

Multiphor II

Buffer Tank

Cooling Plate

Safety lid

IPG Strip Reswelling Tray

Positioning IPG strips on Multiphor

Positioning the electrodes

Cup-loading of Sample

Two - Dimensional Electrophoresis

Principle of 2-D Electrophoresis

1. First dimension:denaturing isoelectric focusing separation according to the pI2. Second dimension:SDS electrophoresis separation according to the MWThe 2-D electrophoresis gel resolves thousands of protein spots.

Common reagents of PAGE

• Monomer: AcrylamideBasic unit in PAGE gelNeurotoxic• Bridge: Bis, [N,N'-methylene-bis(acrylamide)]

Cross-linkerNeurotoxic• Free radical generator: Ammonium persulfate

Generation of free radicalRiboflavin (vitamin B2) can also be used• Catalyst: TEMED (Tetramethylethylenediamine)

Assist transfer of electron of free radical

Choice of electrophoretic system

Choice of electrophoretic system

Second Dimension on Vertical Equipment

Staining Methods

• Colloidal Coomassie stain• Fluorescent stain• Coomassie stain• Silver stain

Sypro Ruby protein staining1. Simple protocol. No overstainng.2. Less protein to protein variation4. Stains glycoproteins, lipoproteins and Ca2+binding proteins and other difficult-to-stain proteins5. Do not stain DNA/RNA6. MS compatible7. Expensive

Staining of Polyacrylamide Gels

References

More help:?? Amersham Pharmacia Biotech Handbook:

On the Internet:http://www.apbiotech.comAngelika Görg’s manual on her Website:http://www.weihenstephan.de/blm/deg