Immunoblotting was performed as described 30 (link). Cells were lysed using cell extraction buffer (Invitrogen, FNN0011) containing protease inhibitor cocktail (Sigma Aldrich, P8340). Total protein was quantified using an EZQ Protein Quantification assay (Invitrogen, R33200). 30 μg of protein were resuspended in 2X reducing sample buffer (Invitrogen, LC2676), electrophoresed on 10-20% tris-glycine gels (Invitrogen), transferred using a semi-dry transfer system (Bio-Rad) to polyvinylidene difluoride membranes (PVDF) (Millipore), and probed with Ms-pAb-anti-CDH19 (Abnova, H00028513-B01P) at 1:250, identified at ∼114k kDa. Rb-pAb-anti-β-actin (Abcam, ab8227) at 1:2000 was used as loading control. Immunocomplexes were detected using luminescence Supersignal West Pico Substrate (Thermo Scientific) per manufacturer instructions.
Protocol detail
Quantitative Western Blotting of CDH19
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FNN0011
protease inhibitor cocktail
semi-dry transfer system
polyvinylidene difluoride membranes (PVDF)
Supersignal West Pico Substrate
Actin
Assay
Buffer
Cell
Gels
Glycine
Luminescence
Membranes
Polyvinylidene difluoride
Protease inhibitor
Protein
Tris
Corresponding Organization :
Other organizations : University of Wisconsin Carbone Cancer Center, University of Wisconsin–Madison
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Variable analysis
independent variables
- Cell lysis using cell extraction buffer containing protease inhibitor cocktail
- Protein quantification using EZQ Protein Quantification assay
- Protein resuspension in 2X reducing sample buffer
- Electrophoresis on 10-20% tris-glycine gels
- Transfer to polyvinylidene difluoride membranes (PVDF)
- Probing with Ms-pAb-anti-CDH19 antibody at 1:250 dilution
- Probing with Rb-pAb-anti-β-actin antibody at 1:2000 dilution
- Detection of CDH19 protein at ~114 kDa
- Detection of β-actin protein (loading control)
- Total protein amount (30 μg) used for immunoblotting
- Rb-pAb-anti-β-actin antibody used as a loading control
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