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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

USE OF IMMOBILIZATION pH GRADIENT (IPG) STRIPS FOR FIRST DIMENSION OF 2D PROTEIN GELS

Use of Immobilization pH Gradient (IPG) Strips for First Dimension of 2D Protein Gels
Contributor: The Electrophoresis Laboratory at Geneva University Hospital
 
Overview
Use of two dimensional gel electrophoresis is a powerful technique to separate hundreds to thousands of proteins from a mixture. The proteins are separated according to their isoelectric points in the first dimension and by mass in the second. The first dimension separation employs an immobilized pH gradient. A standard SDS polyacrylamide gel is used for the second dimension. A non-linear immobilized pH gradient (3.5-10.0 NL IPG 18 cm) was used as the first dimension. It offers high resolution, great reproducibility and allowed high protein loads. Based on the specifications of this protocol, the non-linear pH gradient strips prepared by Amersham Pharmacia Biotechnology AB and are commercially available. Strips should be 3 mm wide and 180 mm long.
 
Procedure
A. Immobilized pH Gradient (IPG) Strip Rehydration

1. Hydration of IPG strips (Amersham Pharmacia) is performed overnight in the reswelling cassette with 25 ml of Hydration Solution.

2. Empty the rehydration cassette.

3. Transfer the IPG strips to the strip tray.

B. Sample Application to Rehydrated IPG Strips

1. Place the IPG strips into the strip tray.

2. Place the IPG strips, humid electrode wicks, electrodes and sample cups into position in the isoelectic focusing apparatus.

3. Cover the IGP strips and ensure that the sample cups are covered with low viscosity paraffin oil.

4. Apply the protein samples at the cathode end of the IPG strips in a slow and continuous manner without touching the gels (see Hint #1).

C. Running Conditions for First Dimension

1. Voltage is linearly increased over 3 hr from 300 V to 3,500 V.

2. Voltage is held constant at 3,500 V for an additional 3 hr.

3. Voltage is then increased to 5,000 V (see Hint #2).

D. IPG Strip Equilibration

1. Incubate the immobilization pH gradient strips in the strip tray for 12 min in 100 ml of Equilibration Buffer #1 (see Hint #3). Discard Equilibration Buffer #1 and add 100 ml of Equilibration Buffer #2 to the strip tray.

2. Incubate the IPG strips in the strip tray for 5 min in Equilibration Buffer #2 to block reduced sulfide bonds.

3. Fractionate the proteins in the second dimension on an SDS Polyacrylamide gel (see Protocol on SDS-PAGE).

Solutions
Equilibration Buffer #2   30% (v/v) Glycerol
50 mM Tris-HCl, pH 6.8
2% (w/v) SDS
0.01% (w/v) Bromophenol Blue
2.5% (w/v) Iodoacetamide
6 M Urea
Equilibration Buffer #1   2% (w/v) DTE
30% (v/v) Glycerol
50 mM Tris-HCl, pH 6.8
2% (w/v) SDS
6 M Urea
Hydration Solution   8 M Urea
2% (w/v) CHAPS
0.01% (w/v) Bromophenol Blue
10 mM Dithioerythritol (DTE)
2% (w/v) Resolyte pH 3.5-10 (Amersham Pharmacia)
 
BioReagents and Chemicals
SDS
Iodoacetamide
Resolyte pH 3.5-10
Dithioerythritol
CHAPS
Urea
Tris
Low Viscosity Paraffin Oil
Immobilized pH Gradient Strips
Glycerol
Bromophenol Blue
 
Protocol Hints
1. The optimal amount of protein sample to load will depend on a variety of factors (i.e. sample is extract versus purified protein) and requires empirical determination to prevent overloading and subsequent loss of resolution.

2. A total volt-hour-product of 100 kVh is used in an overnight run.

3. After the first dimension run, the strips are equilibrated in order to resolubilize the proteins and to reduce disulfide bonds.