Bio-Rad PROTEAN® IEF Cell Accessories - Manual (Page 36)

10.1.2 2 nd Dimension Equilibration Buffers
Prior to running the 2 nd dimension (SDS-PAGE) it is necessary to equilibrate the
ReadyStrips.
The first equilibration step is required to saturate the ReadyStrip IPG Strips with SDS
and reducing agent.
When DTT is used a second equilibration step is required. The Iodoacetamide prevents
protein re-oxidation during electrophoresis and alkylates residual DTT to minimize vertical
streaking.
Equilibration buffer Equilibration Buffer II
(Prepare immediately prior to use) (Prepare immediately prior to use)
Step 1: 10–15 min in equilibration buffer I Step 2 : 10–15 min in equilibration buffer II
6 M Urea 3.6 g/10 ml 6 M Urea 3.6 g/10 ml
2% SDS 0.2 g/10 ml 2% SDS 0.2 g/10 ml
0.375M Tris-HCl 2.5 ml 1.5 M 0.375M Tris-HCl 2.5 ml 1.5 M
pH 8.8 Tris-HCl, pH 8.8 Tris-HCl,
pH 8.8 /10 ml pH 8.8 /10ml
20% Glycerol 2 ml/10 ml 20% Glycerol 2 ml/10 ml
130 mM DTT 200 mg/10 ml 135 mM Iodoacetamide 250 mg/10 ml
10.2 IPG Strip Preparation and Handling
10.2.1. IPG Strip Rehydration
Select IPG strip with the pH range required for optimum separation of the proteins. Refer
to Section 6.2 for IPG strip specifications.
Note: Always wear gloves when handling IPG strips to prevent contamination.
IPG strips must be rehydrated prior to the isoelectric focusing run. Rehydrate the IPG
strips using one of the two basic methods (with or without sample) given below.
Rehydration (with sample)
The recommended method for applying protein samples to ReadyStrip IPG strips is to
rehydrate the strips in buffer containing sample. This is the simplest method to use and
allows for loading of larger quantities of proteins (up to 1 mg) while preventing sample
precipitation. The sample should be prepared in a buffer containing urea, non-ionic and/or
zwitterionic detergents, carrier ampholytes, and a reducing agent. The optimal buffer
composition and protein load will be sample dependent.
Prepare the sample in Rehydration Buffer. In general, use 5–100 µg for silver
staining and up to 1 mg for Coomassie staining. Refer to Section 10.1 for commonly used
rehydration solutions.
Rehydration (without sample)
Some proteins do not diffuse readily into the matrix of the IPG strip. In those cases the
sample can be applied to the IPG strips following rehydration just prior to focusing. The
IPG strips are rehydrated in Rehydration Buffer without sample and the sample is applied
via the channels of the focusing tray.
Select the appropriate disposable rehydration tray or the focusing tray for
rehydration of the IPG strips.
Note: The focusing tray must be used for active rehydration. Passive rehydration of the
IPG strips can take place in the disposable rehydration tray or in the IPG focusing tray.
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