Isolation of Renal Brush Borders

D. James Morré1, Timothy Hammond2

1 Purdue University, West Lafayette, Indiana, 2 Tulane University School of Medicine and Southeast Louisianna Veterans Health Care System, New Orleans, Louisianna
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.26
DOI:  10.1002/0471143030.cb0326s34
Online Posting Date:  March, 2007
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Abstract

Methods are described to isolate intact brush borders and brush border membranes from renal cell homogenates. A rapid method yields sealed vesicles that reconstitute renal brush border transport. In one variation of this protocol, 10 to 20 mM CaCl2 or MgCl2 is added to aggregate non‐brush border structures for subsequent removal by centrifugation. For analytical studies, guidance is provided for subsequent purification steps including preparative free‐flow and aqueous two‐phase partition. Marker enzymes and morphological parameters are included for assessment of yield and fraction purity.

Keywords: kidney; brush border; isolation; aqueous two phase

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Renal Brush Border by Rate Zonal Sucrose Gradients
  • Alternate Protocol 1: Isolation of Kidney Brush Borders by Bivalent Metal Precipitation
  • Alternate Protocol 2: Isolation of Renal Brush Border for Dye/Buffer Uptake
  • Alternate Protocol 3: Isolation of Renal Cortical Membranes Using an Aqueous Two‐Phase Partition Technique
  • Alternate Protocol 4: Incremental Aqueous Two‐Phase Partition Fractionation of Rat Kidney to Yield Both Brush Border and Endosomes
  • Support Protocol 1: Study of Brush Border Membrane Vesicles by Flow Cytometry
  • Support Protocol 2: Analysis of Marker Enzymes for Renal Brush Border
  • Support Protocol 3: Analysis of Renal Brush Border Preparations by Electron Microscopy
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Renal Brush Border by Rate Zonal Sucrose Gradients

  Materials
  • New Zealand white male rabbits (2 to 3 kg)
  • 0.5, 1.4, and 1.7 M sucrose solutions in distilled or deionized water
  • Forceps
  • Scalpel blade or scissors
  • Ground glass homogenizers (hand‐held and motor‐driven e.g., 15‐ml Dounce homogenizer and Potter‐Elvehjem with Teflon pestle, respectively)
  • Polytron homogenizer
  • Phase‐contrast microscope
  • 35‐ml polyallomer ultracentrifuge tubes
  • Refrigerated centrifuge (90,000 × g) with a swinging‐bucket rotor

Alternate Protocol 1: Isolation of Kidney Brush Borders by Bivalent Metal Precipitation

  • 1 M MgCl 2
  • Homogenate of rabbit kidney cortex (prepared from frozen rabbit kidney, see protocol 1, steps 1 to 6)
  • 300 mM mannitol/12 mM HEPES, pH 7.4 (adjusted with Tris)

Alternate Protocol 2: Isolation of Renal Brush Border for Dye/Buffer Uptake

  • Kidneys harvested from one or more (depending on requirements) pentobarbital‐anaesthetized male Sprague‐Dawley rats, 200 to 250 g
  • 20% (w/v) dextran T‐500 (Pharmacia)
  • 40% (w/v) polyethylene glycol 3350 (PEG; Union Carbide or Fisher)
  • 0.2 M potassium phosphate, pH 7.2
  • 30‐ml Pyrex tube
  • Parafilm
  • Pasteur pipets
  • 50‐ml polyallomer tubes
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Figures

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Literature Cited

Literature Cited
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