Glut1 Antibody (A-4): sc-377228

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:1000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

The astrocytes were exposed to 100 µM CoCl2 for the aforementioned periods of time, then the cells were collected and solubilized in lysis buffer (60 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM KCl, 5 mM Na3 EDTA, 3 mM EGTA, and 1% Triton X-100) with protease inhibitors. Following centrifugation at 14,000 × g for 15 min at 4°C, the supernatant was collected and solubilized in loading buffer. Samples were normalized to protein concentration and proteins were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a running voltage of 200 v for 30 min and electrophoretically transferred to polyvinylidene difluoride membranes at 100 V for 1 h. The membranes were blocked with 5.0% fat-free milk in PBS with 0.05% Tween-20 (PBST) for 1 h at room temperature, and incubated separately with mouse monoclonal anti-GLUT-1 (cat. no. sc-377228; dilution 1:1,000), rabbit GLUT-3 (cat. no. sc-74399; dilution 1:1,000), mouse monoclonal anti-HIF-1α (cat. no. sc-71247; dilution 1:1,000) or mouse β-actin (cat. no. sc-47778; dilution 1:2,000; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Subsequently, the membranes were washed with PBST three times for 10 min each. Following incubation with Alexa Fluor® 488 donkey anti-rabbit IgG (cat. no. R37118) and Alexa Fluor® 680 rabbit anti-mouse IgG (cat. no. A-21065; Thermo Fisher Scientific, Inc.; dilution 1:10,000) for 60 min at room temperature in the dark, blots were detected on an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and relative density units were estimated from the mean pixel density using Image Studio Lite version 4.0 (LI-COR Biosciences) and normalized to β-actin. Data are presented as ratios over control (time point: 0 h).

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