Total plasma membrane protein was harvested using ultracentrifugation according to Zhang et al. (30 (link)). For tumor samples, tumors were thawed on ice and homogenized in lysis buffer (29 (link)), supplemented with protease inhibitors (20 mL/g tumor). Tumor homogenates were centrifuged at 12,000 rpm for 10 min at 4°C and the resulting supernatants were collected for Western blot analysis. The Western blotting (20 μg total protein per lane) was performed as previously described (29 (link)) using the following antibodies: MCT1 (1:5000; ab3538P, EMD Millipore), MCT4 (1:500; sc50329, Santa Cruz), CD147 (1:100; sc9757, 1:1000; sc9753, Santa Cruz), GLUT1 (1:500; ab40084), the proliferation biomarker Ki67 (1:500; sc23900, Santa Cruz), and GAPDH (1:5000; sc25778, Santa Cruz).
Protocol detail
Plasma Membrane Protein Extraction and Western Blot Analysis
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Antibodies
Biomarker
Buffer
Cd147
Gapdh
Glut1
Mct1
Membrane
Membrane protein
Plasma
Plasma membrane
Plasma protein
Protease inhibitors
Protein
Tumor
Ultracentrifugation
Western blot analysis
Corresponding Organization :
Other organizations : University at Buffalo, State University of New York
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Variable analysis
independent variables
- Tumor samples were thawed on ice and homogenized in lysis buffer, supplemented with protease inhibitors (20 mL/g tumor).
- Total plasma membrane protein was harvested using ultracentrifugation.
- Western blot analysis was performed to measure the levels of MCT1, MCT4, CD147, GLUT1, the proliferation biomarker Ki67, and GAPDH.
- Centrifugation was performed at 12,000 rpm for 10 min at 4°C.
- Protein loading for Western blotting was 20 μg total protein per lane.
- No positive or negative controls were explicitly mentioned.
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