Abstract
Abstract A method for isolating brush borders from rat kidney homogenates has been developed. Minced tissue, 27 g, is gently homogenized in 20 mm sodium or potassium bicarbonate, pH 8.1. The diluted homogenate, containing large fragments of brush borders, is filtered and then fractionated with the use of combined differential, isopycnic, and rate-zonal centrifugations. The purity of the final brush border fraction is documented with both morphological and enzymatic studies. Electron micrographs of the final brush border fraction show microvilli profiles with few cytoplasmic contaminants. Alkaline phosphatase assays show a 15-fold increase in specific activity of the brush border fraction relative to the starting homogenate. Acid phosphatase, glucose 6-phosphatase, and succinate cytochrome c reductase studies are consistent with contamination rates of 0.04% for lysosomes, 0.2% for microsomes, and 7% for mitochondria. Calculations based on recovery of total alkaline phosphatase activity indicate that the procedure recovers 21% of the alkaline phosphatase present in the starting homogenate. The final brush border pellet obtained from 27 g of whole kidney has a wet weight of 0.27 g and a protein content of 20 mg.
Highlights
A method for isolating brush borders from rat kidney homogenateshas been developed
High resolution electron microscopy performed on multiple samplestaken from the final brush border fraction showedwell preservedmicrovilli with basalmembranecontinuity
We have described a procedure for isolating a brush border fraction from rat kidney
Summary
Step I-Of the above40.9% solution, ml werepouredintoMaterials eachof three SW-25.1tubesand carefully overlayed with 1.0ml of a freshsolutioncontaining40.4 f 0.1y0sucrose.Water was de-ionized, and sucrosewas reagent grade. All Step J-Centrifugation at 90,000X gmax(25,000rpm) for 75 sucroseconcentrations, which are reported as percentage of min (brakeon) resultedin a thin, light brownfloating layer and a weight by weight, were determinedwith a Bauschand Lomb dark brown pellet. Abbe 3L refractometer by reading the “total dissolvedsolids” Step K-The floats were removed with a Spoonula (Fisher scaleat room temperatureand correctingto 20” (International 14-375-10a) nd addedto 10ml of Medium C. Homogenizationswere performedin a Step L-The resuspendedfloating layers were centrifuged at largeDounceglasshomogenizer(BlaessigGlass,Rochester,New 3000 x gmaxfor 5 min in an anglehead rotor Media referred to in the procedurewereasfollows: Medium A, 20 mMsodiumor potassiumbicarbonate, pH. 8.1; Medium B, 10 mM sodiumor potassiumbicarbonate, pH. 8.1; Medium C, 4 mMsodiumor potassiumbicarbonateand 1mM magnesiumchloride, pH 8.1. Homogenization was completedwith four upward gentle strokes of the loose pestle.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
