Carbonate Extraction of Cell Membrane Proteins
Sample concentration
Standard format for 2-D analysis is a 20 cm x 20 cm and 1.5 mm thick gel, requiring an 18 cm immobilized pH gradient (IPG) strip (Amersham Pharmacia Biotech). Typically 1-3 mg of a complex protein mixture is needed per 2-D gel for staining by Coomassie Blue. Silver stain and fluorescence staining are more sensitive and are available for low-level samples. Each gel requires 100-300mg of sample.
Protein estimations are based on the following generalizations. Whole cell pellets are estimated to be ca. 7% protein. Membrane proteins are estimated at 10% of the total protein or 0.7% of the whole cell pellet. Wet weight of whole cell pellet is estimated from packed cell volume. Assume a density of 1 g/ml.
Preparation
Cell pellets are resuspended with 100 ml of 50 mM Tris/HCl, pH 7.3 containing 0.1 mg/ml of DNase I per 50 mg cell wet weight. The samples are kept on ice and sonicated on low power setting with five 3-4 second bursts with 1 min between bursts. Unbroken cells are removed by centrifugation at 2,500 g for 10 min. Following centrifugation, the supernatant is diluted 10 fold with ice cold 0.1 M sodium carbonate, pH 11 and stirred slowly on ice for 1 hr. The carbonate treated membranes are collected by ultracentrifugation at 100,000 g for 1 hr at 4°C. The resulting pellet in resuspended in 50 mM Tris/HCl pH 7.3 and centrifuged for 20 min at 115,000 g at 4°C. The washed pellet can then be solubilized for 2-D analysis.
Solutions
50 mM Tris/HCl, pH 7.3
100 mM sodium carbonate, pH 11
Dnase stock solution:
DNase I, 1 mg/ml
500 mM Tris/HCl, pH 7.0
50 mM MgCl2
References
Fujiki Y, Hubbard AL, Fowler S, Lazarow PB. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97-102.
Molloy MP, Herbert BR, Slade MB, Rabilloud T, Nouwens AS, Williams KL, Gooley AA. Proteomic analysis of the Escherichia coli outer membrane. Eur J Biochem. 2000 May;267(10):2871-81.
Current Protocols in Protein Science. (2003) Coligan JE, et al., Eds. John Wiley & Sons, Inc. New York.
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Protocols
The Michigan Proteome Consortium follows a standard set of protocols listed below:
Whole cell lysate
Membrane proteins
IPG 1st dimension
SDS page
Colloidal coomassie stain
Fluorescence stain
Silver stain
In-gel digestion
References and suggested reading
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