The ReadyPrep Protein Extraction Kit (Membrane II) is optimized for the isolation of proteins containing more than two transmembrane domains and proteins that are resistant to extraction by the ReadyPrep Membrane I Kit.

• Isolates complex proteins with 3 or more transmembrane domains and those resistant to extraction by the ReadyPrep™ Protein Extraction (Membrane I) Kit
• Useful for a wide variety of biological samples, including animal tissue, animal cells, plant tissue, yeast, and bacteria
• Requires ultracentrifugation; entire procedure can be completed in less than 3 hours
• Includes a strongly chaotropic, IEF-compatible rehydration/sample buffer that contains the detergent ASB-14 to efficiently solubilize proteins

 
The ReadyPrep Protein Extraction Kit (Membrane II) is based on the method of Fujiki et al. (1982) for the isolation of cellular membrane fractions after carbonate extraction, and the later adaption of the technique by Molloy et al. (2000). Incubation of tissue or cells with the high-pH Membrane Protein Concentrating Reagent strips weakly associated proteins away from membrane fragments. After ultracentrifugation and washing, the membrane pellet is solubilized using an IEF buffer or other buffer. The included 2-D Rehydration/Sample Buffer 1 contains the zwitterionic detergent ASB-14. The kit also contains the reducing agent tributylphosphine (TBP), which is more efficient than dithiothreitol (DTT) for keeping proteins reduced in 2-D PAGE applications.

Kit Components

For 10 extractions of 100–200 mg of cells or tissue:

• 2-D Rehydration/Sample Buffer 1, 2 vials, reconstituted total 10 ml
• Lysis Buffer, reconstituted 50 ml
• Membrane Protein Concentrating Reagent, reconstituted 125 ml
• TBP Reducing Agent, 1 ampoule; 0.6 ml of 200 mM tributylphosphine (TBP) in 1-methyl-2-pyrrolidinone sealed under nitrogen gas
• Empty vial for TBP storage
• Instruction manual

Bio-Rad offers a range of ReadyPrep Kits for Fractionation by Cellular Location. These kits reduce sample complexity, increase detection and identification of low-abundance proteins, and simplify proteomic studies.

Related Products

• 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
• ReadyPrep Reduction-Alkylation Kit (1632090)
• 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 Protein Extraction Kit (Membrane II) 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

Fujiki Y et al. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biology 93, 97–102. PMID: 7068762
Molloy MP et al. (2000). Proteomic analysis of the Escherichia coli outer membrane. Eur J Biochem 267, 2871–2881. PMID: 10806384