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Dr. A. Khaleel AhamedAssociate Professor of Botany
Jamal Mohamed CollegeTiruchirappalli – 620 020
[email protected] 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