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Nevada Proteomics Center

Phone(775) 784-6337: 2-D Gels
Phone(775) 784-1590: Mass Spectrometry
Fax(775) 784-1419
Emailquilici@unr.edu
Location School of Medicine
Manville Medical Building, Rooms 15-17, MS 0330
Address 1664 N. Virginia Street
Reno,  NV  89557-MS 0330
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Protocol: Preparation of Soluble and Membrane Protein Fractions

The following method will isolate membranes of all types from tissue. It has been optimized for use with mammalian tissues. Other sample types may require a more severe method for initial breakage of cells.

1. Prepare a homogenate

For mammalian tissue:

  • Mince tissue into small pieces using a very clean scalpel. Transfer the pieces to a Potter-Elvehjem homogenizer on ice.
  • Add 1 ml cold homogenization buffer (250 mM sucrose, 1 mM EDTA, 10 mM Tris HCl buffer, pH 7.2 plus protease inhibitors) to the homogenizer. Homogenize the tissue using approximately 20 manual up and down strokes. Transfer samples to microfuge tubes.
  • Sonicate sample using two 10 second pulses (30 seconds in between pulses) using a probe sonicator. Keep samples in an ice bath and keep probe away from the sample-air interface to minimize foaming.

For pellets of cells or tiny amounts of tissue:

  • Add 1 ml cold homogenization buffer (250 mM sucrose, 1 mM EDTA, 10 mM Tris HCl buffer, pH 7.2 plus protease inhibitors) to the pellet of cells or tissue in the bottom of a 1.5 ml microfuge tube. Homogenize the tissue using a Teflon pestle intended for use in such tubes. Homogenize using approximately 20 manual up and down strokes. Transfer samples to microfuge tubes.
  • Sonicate sample using two 10 second pulses (30 seconds in between pulses) using a probe sonicator. Keep samples in an ice bath and keep probe away from the sample-air interface to minimize foaming.

For tougher tissues:

  • If there is sufficient sample, prepare a nitrogen powder from the sample. Add the sample to cold homogenization buffer (250 mM sucrose, 1 mM EDTA, 10 mM Tris HCl buffer, pH 7.2 plus protease inhibitors) to the homogenizer. Homogenize the tissue using approximately 20 manual up and down strokes. Transfer samples to microfuge tubes.
  • Sonicate sample using two 10 second pulses (30 seconds in between pulses) using a probe sonicator. Keep samples in an ice bath and keep probe away from the sample-air interface to minimize foaming.
  • If there is not sufficient sample to prepare a nitrogen powder, then cut the sample up with a clean scalpel, place in homogenization buffer and use a tissue tearer-type device to disrupt the sample.

2. Separate Soluble Proteins from Membranes

  • Remove intact cells, nuclei and cell debris by centrifugation of the homogenate at 500 x g for 10 minutes at 4°C (This step may be repeated if necessary). Discard pellet.
  • Centrifuge the supernatant at 100,000 x g for 1 hour at 4°C.
  • Remove the supernatant. This contains soluble proteins. They may be precipitated by any protein precipitation method. For separation using 2-D gels, dissolve the protein precipitate in any standard rehydration buffer. For LC/MS, the precipitate should be dissolved in 50 µl 25 mM ammonium bicarbonate.
  • Wash the pellet with homogenization buffer and recentrifuge at 100,000 x g, 4°C, for 1 hour. Discard the supernatant. The pellet contains all of the cell's membrane fraction.
  • For protein separation using 2-D gels, extract proteins from the membrane pellet using a rehydration buffer with strong detergents, such as Bio-Rad's Sequential Extraction Reagent 3 or a solution of 7 M urea, 2 M thiourea, 2% ASB-14 (amidosulfobetaine 14). Allow the pellet to incubate with the buffer for at least one hour with occasional vortexing and then centrifuge at 100,000 x g, 4°C, for 1 hour. Remove and save the supernatant.
  • The most effective method of solubilizing and visualizing membrane proteins that are to be identified by LC/MS is to extract the proteins from the membrane using a minimum amount (50 µl, if possible) of Laemmli buffer with β-mercaptoethanol. Centrifuge at 100,000 x g, 4°C, for 1 hour. Apply the supernatant to a standard 1-D gel. The gel may then be run until the dye front reaches the bottom of the gel, to separate the membrane proteins, or may be run only until the dye front completely clears the well. This latter method concentrates all membrane proteins in one band. The band may be cut out and submitted for trypsin digestion.

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University of Nevada, Reno
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Reno,  NV  89557-

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