Protein lysates were prepared from mouse tissue employing MAP Kinase lysis buffer as previously described(Lanaspa et al., 2007 (link)). Protein content was determined by the BCA protein assay (Pierce). Total protein (50 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% w/v), and transferred to PVDF membranes (BioRad). Membranes were first blocked for 1 h at 25 °C in 4% (w/v) instant milk dissolved in 0.1 % Tween-20 Tris-Buffered Saline (TTBS), incubated with primary rabbit or mouse-raised antibodies (1:1000 dilution in TTBS) KHK (Sigma HPA007040; RRID:AB_1079185), KHK-A (SAB Signalway; Cat# 21708), KHK-C (SAB Signalway; Cat# 21709), Glut5 (Millipore, 07–1406 lot 2999837, this antibody and lot has been validated using specific Glut5 KO mice, a representative western blot is shown in supplemental figure 2 ) and Actin (Cell Signaling 4968; RRID:2313904) and visualized using an anti-rabbit (7074; RRID:AB_2099233) or anti-mouse IgG (7076; RRID:AB_330924) horseradish-peroxidase conjugated secondary antibody (1:2000, Cell Signaling) using the HRP Immunstar® detection kit (Bio-Rad, Hercules, CA). Chemiluminescence was recorded with an Image Station 440CF and results analyzed with the 1D Image Software (Kodak Digital Science, Rochester, NY).
Glut5
Manufactured by Merck Group
Sourced in United States
Glut5 is a laboratory equipment product developed by Merck Group. It is a high-performance device designed for the measurement and analysis of glucose levels. The core function of Glut5 is to provide accurate and reliable glucose detection capabilities in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (2 mentions)
Image station 440cf, by Kodak (2 mentions)
1d image software, by Kodak (2 mentions)
Glut5, by Cell Signaling Technology (1 mentions)
Hrp immunstar detection kit, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
2 protocols using glut5
1
Western Blot Analysis of Metabolic Enzymes
2
Western Blot Analysis of Mouse Tissue Proteins
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Protein lysates were prepared from mouse tissue using MAPK lysis buffer as previously described [18 (link)]. Protein content was determined by the BCA protein assay (Pierce, Rockford, IL, USA). Total protein (50 μg) was separated by SDS-PAGE [10% (wt/vol)] and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were first blocked for 1 h at 25 °C in 4% (wt/vol) instant milk dissolved in 0.1% Tris-buffered saline with Tween 20 TBS (TTBS) and incubated with the following primary rabbit- or mouse-raised antibodies (1:1000 dilution in TTBS): KHK (HPA007040, Sigma, St. Louis, MO, USA), Glut5 (07-1406, Millipore, Burlington, MA, USA), and actin (no. 3700, Cell Signaling) and visualized using an anti-rabbit (no. 7074) or anti-mouse IgG (no. 7076) horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, Cell Signaling) using the SuperSignal West Pico PLUS (34577, ThermoFisher Scientific). Chemiluminescence was recorded with an Image Station 440CF, and results were analyzed with the 1D Image software (Kodak Digital Science, Rochester, NY, USA). Data for proteins of interest are normalized to β-actin expression.
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