Isolation of Intestinal Brush‐Border Membranes

Chris I Cheeseman1, Debbie O'Neill1

1 University of Alberta, Edmonton, Alberta
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.21
DOI:  10.1002/0471143030.cb0321s30
Online Posting Date:  April, 2006
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Abstract

This unit provides protocols for isolating intestinal brush‐border membranes from rat, pig, and cow. These membranes can be used for immunoblotting or other analytical techniques. They will also spontaneously form closed vesicles which allow for flux assays to be performed using rapid filtration techniques. Overall the isolation procedures take ∼3.5 hr. The resulting isolated membranes can be stored under liquid nitrogen or at −70°C for a week or more depending upon the species.

Keywords: intestinal brush‐border membrane; membrane vesicles; centrifugation

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Intestinal Brush‐Border Membranes from Rat
  • Support Protocol 1: Assessment of Membrane Enrichment
  • Alternate Protocol 1: Isolation of Intestinal Brush‐Border Membranes from Pig
  • Alternate Protocol 2: Isolation of Intestinal Brush‐Border Membranes from Cow
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Intestinal Brush‐Border Membranes from Rat

  Materials
  • Anesthetic (e.g., sodium pentobarbital; MTC Pharmaceuticals, Ontario, Canada)
  • Rats (e.g., male Sprague‐Dawley rats, ∼300 g)
  • Isotonic saline (0.9% w/v NaCl) containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF), ice‐cold
  • Water, ice cold
  • Rat collection solution A (see recipe)
  • Formaldehyde
  • 100 mM MgCl 2
  • Rat homogenization solution B (see recipe)
  • Final suspension medium (see recipe)
  • 1‐ml syringe with 26‐G needle
  • Surgical instruments for laproscopic surgery: scissors and forceps
  • 30 × 20–cm Plexiglas or other flat plate, chilled on ice
  • Glass microscope slides, chilled on ice
  • 100‐ and 250‐ ml beakers
  • Polytron homogenizer (Brinkmann) or equivalent
  • Magnetic stirrer and stir bar
  • 50‐ml polycarbonate centrifuge tube
  • High‐speed centrifuge capable of generating 20,000 × g and handling samples up to 50 ml
  • 15‐ml hand held glass homogenizer
  • Additional materials for determination of protein concentration ( appendix 3H) and fixing, sectioning, and staining tissue for light microscopy (optional)

Support Protocol 1: Assessment of Membrane Enrichment

  • Pig intestine (freshly isolated from slaughter or animal house)
  • Pig and cow collection solution A, ice cold
  • Pig and cow homogenization solution B
  • 25‐G needle
  • 10‐ml syringe
  • −70°C freezer

Alternate Protocol 1: Isolation of Intestinal Brush‐Border Membranes from Pig

  • Cow intestine
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 0.1 mM PMSF and 0.1 mM aprotonin
  • Pig and cow collection solution A (see recipe)
  • Pig and cow homogenization solution B (see recipe)
  • Aluminum foil or plastic vials suitable for freezing
  • Liquid nitrogen or −70°C freezer
  • 25‐G needle
  • 10‐ml syringe
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Figures

Videos

Literature Cited

Literature Cited
   Fredricksen, B. and Wirsen, C. 1956. In‐vivo effect of colchicine on alkaline phosphatase of rat intestinal epithelium. Exp. Cell Res. 10:749‐751.
   Hauser, H. and Semenza, G. 1983. Sucrase‐isomaltase: A stalked intrinsic protein of the brush border membrane. CRC Crit. Rev. Biochem. 14:319‐345.
   Hearn, P.R., Russell, R.G., and Farmer, J. 1981. The formation and orientation of brush‐border vesicles from rat duodenal mucosa. J. Cell Sci. 47:227‐236.
   Hopfer, U., Nelson, K., Perrotto, J., and Isselbacher, K.J. 1973. Glucose transport in isolated brush border membrane from rat small intestine. J. Biol. Chem. 248:25‐32.
   Maenz, D.D., Chenu, C., Bellemare, F., and Berteloot, A. 1991. Improved stability of rabbit and rat intestinal brush border membrane vesicles using phospholipase inhibitors. Biochim. Biophys. Acta 1069:250‐258.
   Prabhu, R. and Balasubramanian, K.A. 2001. A novel method of preparation of small intestinal brush‐border membrane vesicles by polyethylene glycol precipitation. Anal. Biochem. 289:157‐161.
   Tsuboi, K.K., Kwong, L.K., Yamada, K., Sunshine, P., and Koldovsky, O. 1985. Nature of elevated rat intestinal carbohydrase activities after high‐carbohydrate diet feeding. Am. J. Physiol. 249:G510‐G518.
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