Choose the ReadyPrep Reduction-Alkylation Kit to reduce disulfide bonds and block their formation in isoelectric focusing (IEF) samples. The kit contains tributylphosphine (TBP) to reduce both intramolecular and intermolecular disulfide bonds, and iodoacetamide to then alkylate the thiols.

• Improve IEF resolution and reproducibility
• Eliminate disulfide bond formation during IEF
• Obtain a consistent spot pattern, and minimize streaking and aberrant spot migration
• Use directly in rehydration/sample buffer; complete in less than 2 hours, with very little hands-on time
• Compatible with other ReadyPrep sample preparation kits and buffers

The ReadyPrep Reduction-Alkylation Kit is a simple and effective method to improve the results of 2-D electrophoresis. Many reducing agents become negatively charged during isoelectric focusing and migrate off the IPG strip.  Furthermore, dithiothreitol (DTT), the most commonly used reducing agent, is not effective under acidic conditions. TBP has been shown to be more effective reducing agent than DTT for 2-D gel electrophoresis. Additionally, alkylation improves the resolution of basic proteins, which have an increased rate of disulfide bond formation in an alkaline environment (Görg et al. 2000, Hoving et al. 2002).

The combination of reduction with TBP and the subsequent alkylation of the thiols with iodoacetamide is effective in reducing intramolecular and intermolecular disulfide bonds and preventing the reformation of disulfide bonds and disulfide shuffling, resulting in higher resolution, less spot ambiguity, and increased reproducibility.

Kit Components

For treatment of 100 samples, each 300 µl containing up to 10 mg/ml protein

• TBP (200 mM), 0.6 ml, 3 ampoules
• Empty glass vials for TBP storage after opening the ampoules, 3
• Iodoacetamide, 56 mg, 5 amber vials
• Alkylation buffer, 2 ml
• Instruction manual

Related Products

• ReadyPrep Kits for Fractionation by Cellular Location
• ReadyPrep Kits for Fractionation by Solubility
• Aurum™ Ion Exchange Mini Kits for Fractionation by Charge
• Rotofor^® Liquid-Phase IEF Cells for Fractionation by pI
• Model 491 Prep Cell and Mini Prep Cell for Fractionation by Solubility
• ReadyPrep™ 2-D Starter Kit
• PROTEAN^® i12™ IEF System
• ReadyStrip™ IPG Strips
• PowerPac™ HV High-Voltage Power Supply

Related Categories

• Protein Gels
• Electrophoresis Chambers
• 2-D Electrophoresis Buffers and Reagents
• Ampholytes
• Tube Gel IEF Systems
• Gel Imaging Systems
• 2-D Electrophoresis Workflow

Supporting Documentation

• ReadyPrep Reduction-Alkylation Kit Instruction Manual
• Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure
• ReadyStrip IPG Strip Instruction Manual
• PROTEAN i12 IEF System Brochure
• Host Cell Protein (HCP) Workflow Quick Guide

References

Görg A et al. (2000). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053. PMID: 10786879
Hoving S et al. (2002). Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2, 127. PMID: 11840558