Reaktion #2045061

Reaktionsgleichung

X-100

leupeptin

pepstatin

Tris-HCl

sodium pyrophosphate

EDTA

DTT

phenylmethylsulfonyl fluoride

glycerol

→

Glucose

Reaktionsbedingungen

Aufarbeitung

1. 1
   SonstigeFresh intact proximal jejunum was removed
2. 2
   Waschenwashed with saline
3. 3
   Sonstigewere removed
4. 4
   workup.ADDITIONcontaining 50 mM mannitol and protease inhibitors (5 μM aprotinin, leupeptin, and pepstatin)
5. 5
   workup.ADDITIONPEG 4000 was added to a final concentration of 10%
6. 6
   workup.STIRRINGstirred on ice for 15 minutes
7. 7
   workup.WAITg for 60 minutes at 4° C
8. 8
   WaschenThe pellet was washed in suspension buffer (10 mM Tris-HCl, pH 7.1
9. 9
   workup.ADDITIONcontaining 300 mM mannitol and protease inhibitors 5 μM aprotinin
10. 10
    Sonstigeleupeptin, and pepstatin) and collected again by centrifugation for 5 minutes
11. 11
    Sonstigeg at 4° C
12. 12
    SonstigeFor preparation of total membranes
13. 13
    Temperaturfrozen jejunum sections (1 g)
14. 14
    workup.WAITg for 20 minutes at 4° C.
15. 15
    Sonstigeto remove insoluble material

Vorschrift

Fresh intact proximal jejunum was removed, washed with saline and placed on ice while approximately 4 g of mucosa were removed and transferred to cold 2 mM Tris-HCl buffer (pH 7.1) containing 50 mM mannitol and protease inhibitors (5 μM aprotinin, leupeptin, and pepstatin). The mucosa was then homogenized and PEG 4000 was added to a final concentration of 10% and stirred on ice for 15 minutes. The homogenate was then centrifuged for 15 minutes at 7,500×g and the resulting supernatant fraction centrifuged at 27,000×g for 60 minutes at 4° C. The pellet was washed in suspension buffer (10 mM Tris-HCl, pH 7.1, containing 300 mM mannitol and protease inhibitors 5 μM aprotinin, leupeptin, and pepstatin) and collected again by centrifugation for 5 minutes, 27,000×g at 4° C. The crude brush border membrane (BBM) pellet was suspended in 1 mL of suspension buffer. For preparation of total membranes, frozen jejunum sections (1 g) were and homogenized on ice in 700 μL Buffer A (50 mM Tris-HCl pH 7.5, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol) containing 1% Triton X-100 and 5 μM aprotinin, leupeptin, and pepstatin. The homogenates were centrifuged at 6,000×g for 20 minutes at 4° C. to remove insoluble material. The protein concentrations of the total and BBM preparations were determined using BCA reagents (Pierce, Rockford, Ill. USA). Final total and brush border membrane preparations were frozen at −80° C. until assayed. The purity of the brush border membrane preparations as measured by alkaline phosphatase were not affected by treatment (data not shown).

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