Abstract
Twodimensional gel electrophoresis may be the mixture of two highresolution electrophoretic procedures (isoelectric focusing and SDSpolyacrylamide gel electrophoresis) to supply much greater resolution than either procedure alone. Within the firstdimension gel, solubilized proteins are separated based on their isoelectric point (pI) by isoelectric focusing. This gel will be applied to the peak of the SDSslab gel and electrophoresed. The proteins within the firstdimension gel migrate in to the seconddimension gel where they're separated based on their molecular weight. The fundamental protocols within this unit derive from the kind of equipment initially explained O'Farrell in 1975. For very fundamental or very acidic proteins, two alternate protocols are supplied. Another alternate protocol describes how twodimensional electrophoresis can be carried out utilizing a minigel system. Protein sample preparation is presented within the support protocol.
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Table of Contents
- Fundamental Protocol 1: First‐Dimension (Isoelectric‐Focusing) Gels
- Support Protocol 1: Solubilization and Preparation of Proteins in Tissue Samples
- Fundamental Protocol 2: Second‐Dimension Gels
- Alternate Protocol 1: Isoelectric Focusing of Very Fundamental Proteins Using Nephge
- Alternate Protocol 2: Isoelectric Focusing of Very Acidic Proteins Using Nephge
- Alternate Protocol 3: Two‐Dimensional Minigels
- Alternate Protocol 4: Two‐Dimensional (2‐D) Electrophoresis with Immobilized pH Gradients
- Support Protocol 2: Rehydration of IPG Strips While using Immobiline Drystrip Reswelling Tray
- Reagents and Solutions
- Commentary
- Literature Reported
- Figures
- Tables
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Materials
30% acrylamide/1.8% bisacrylamide (see recipe )
Ampholytes, pH 4 to eight (Bio‐Rad, Serva, Invitrogen, Sigma‐Aldrich)
Nonidet P (NP)
TEMED ( N. N. N ′, N ′ tetramethylethylenediamine)
10% ammonium persulfate (see recipe )
.085% phosphoric acidity (see recipe )
.02 M NaOH (see recipe )
Protein samples (see protocol 2 )
Concentrated bromphenol blue (see recipe )
Chromic acidity cleaning solution (see recipe )
1.‐ to three.‐mm‐inner‐diameter glass gel tubes (.5 in. more than the width from the second‐dimension gel 4‐ to six‐mm outer diameter)
2.5‐ to three.‐cm‐inner‐diameter gel‐casting glass tube, cm shorter than gel tubes
Small vacuum flask
50‐µl, 1‐ml, and 20‐ml syringes
.2‐ or .45‐µm filter capsule (Acrodisk Gelman)
Single‐edge blade
Tube cell (Bio‐Rad, Topac, CBS Scientific, Scie‐Plas)
22‐G hypodermic needle (2‐in. lengthy)
200‐µl pipettor tip
1‐dram gel vials
SDS or urea solubilization buffer (see reciperecipes )
Dounce homogenizer with pestles A and B
200‐µl centrifuge tubes
Beckman 42.2‐Ti rotor (or equivalent)
30% acrylamide/.8% bisacrylamide (see recipe for acrylamide/bisacrylamide solutions)
Gel buffer (see recipe )
10% ammonium persulfate (see recipe )
Isobutyl alcohol, H 2 O‐saturated
Stacking gel buffer (optional see recipe )
First‐dimension gel (see protocol 1 )
Equilibration buffer (see recipe )
Hot .5% and 1% agarose (see recipe retain in boiling water bath)
Protein molecular weight standards (Table 97.80.4711 like kits Bio‐Rad or Amersham Biosciences)
SDS solubilization buffer (see recipe )
Reservoir buffer (see recipe ), prechilled to 10° to twenty°C
Coolant (from running plain tap water or circulating refrigerated water bath)
Gel plates, one lengthy and something short
1.5‐mm spacers ( cm × 14 cm × .75 mm)
Gel identification tag (e.g. typed consecutive figures on filter paper)
5 × 15cm glass plate
PROTEAN II electrophoresis cell (Bio‐Rad)
Ampholytes, pH 2 to 11 (Serva)
.01 M phosphoric acidity, deaerated
4 M urea, deaerated
To evaluate very fundamental proteins, the process is equivalent to described in protocol 1 for first‐dimension gels using the following exceptions within the indicated steps:
Ampholytes, pH 2.5 to 4 (Pharmacia LKB)
Concentrated sulfuric acidity
Protein sample for analysis
Sample solution: default sample solution, hydrophobic sample solution, or tissue sample solution (see recipe )
Tissue homogenization solution (see recipe )
Rehydration stock solution (see recipe )
IPG buffer or Pharmalyte (same range because the IPG strip Amersham Biosciences)
Cleaning solution (e.g. Ettan IPGphor Strip Holder Cleaning Solution Amersham Biosciences)
IPG Dry Strip Cover Fluid (Amersham Biosciences)
Vertical gel for SDS‐PAGE ( protocol 3 in unit 10.2 )
SDS equilibration buffer (see recipe )
SDS electrophoresis buffer (unit 10.2 )
Molecular weight markers (unit 10.2 )
Agarose sealing solution (see recipe )
Isoelectric focusing system (Ettan IPGphor, Amersham Biosciences PROTEAN IEF System, Bio‐Rad)
IPG strips (Amersham Biosciences)
100°C heating block
Thin plastic ruler
Additional reagents and equipment for second‐dimension gel electrophoresis (see protocol 3 )
Immobiline DryStrip Reswelling Tray (Amersham Biosciences)
