Proteins from HCT116 cells were extracted by using RIPA buffer. BCA assay was utilized to measure protein concentration. The extracted proteins were subjected to 10% SDS/PAGE and transferred onto 0.22 µm PVDF membranes (REF 03010040; Roche, Pleasanton, CA, U.S.A.). The membranes were blocked with 5% nonfat milk and incubated with anti-β-catenin, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-p65, anti-IκBα, anti-histone H1, anti-tubulin (all from Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) and anti-GAPDH antibodies (Abcam, Cambridge, MA, U.S.A.) overnight at 4°C. After washing five times (6 min each time), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam) for 1 h at room temperature. The bands were visualized by using an enhanced chemiluminescent kit (Thermo Fisher Scientific). Immunopositive bands were detected using a FluorChem M system (ProteinSimple, San Jose, CA, U.S.A.).
0.22 m pvdf membranes
Manufactured by Roche
Sourced in United States
The 0.22 μm PVDF membranes are a type of lab equipment used for filtration purposes. These membranes are made from polyvinylidene fluoride (PVDF) and have a pore size of 0.22 micrometers, allowing for the separation and retention of particles, cells, or other materials during filtration processes.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescent kit, by Thermo Fisher Scientific (1 mentions)
Anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti iκbα, by Santa Cruz Biotechnology (1 mentions)
Anti histone h1, by Santa Cruz Biotechnology (1 mentions)
Anti p65, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Anti tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (1 mentions)
Anti n cadherin, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence solution, by Merck Group (1 mentions)
2 protocols using 0.22 m pvdf membranes
1
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
2
Western Blot and Immunofluorescence Assay
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Total cell protein was separated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) then transferred to 0.22 µm PVDF membranes
(Roche). The membranes were incubated with 5% non-fat milk for 2 h at
room temperature to block nonspecific binding. The respective specific
primary antibody was then incubated on the membranes at 4°C overnight.
The following primary antibodies were used: GAPDH (1:2000, CST,
Beverly, MA, USA), E-cadherin (1:1000, CST), N-cadherin (1:1000, CST),
vimentin (1:1000, CST), ZEB2 (1:500, Millipore). For
immunofluorescence, cells were plated on 24-well plate and stained
using E-cadherin (1:500, CST), and N-cadherin (1:500, CST). After
washing 3 times with PBST, horseradish peroxidase (HRP)-conjugated
secondary antibodies were incubated on the membranes at 25°C for 1 h.
GAPDH was used as the internal control. All western blot experiments
were replicated 3 times. The membranes were then stained by enhanced
chemiluminescence solution (Millipore). Nuclei were visualized by
co-staining with DAPI and data was imaged with an Olympus BX63.
electrophoresis (SDS-PAGE) then transferred to 0.22 µm PVDF membranes
(Roche). The membranes were incubated with 5% non-fat milk for 2 h at
room temperature to block nonspecific binding. The respective specific
primary antibody was then incubated on the membranes at 4°C overnight.
The following primary antibodies were used: GAPDH (1:2000, CST,
Beverly, MA, USA), E-cadherin (1:1000, CST), N-cadherin (1:1000, CST),
vimentin (1:1000, CST), ZEB2 (1:500, Millipore). For
immunofluorescence, cells were plated on 24-well plate and stained
using E-cadherin (1:500, CST), and N-cadherin (1:500, CST). After
washing 3 times with PBST, horseradish peroxidase (HRP)-conjugated
secondary antibodies were incubated on the membranes at 25°C for 1 h.
GAPDH was used as the internal control. All western blot experiments
were replicated 3 times. The membranes were then stained by enhanced
chemiluminescence solution (Millipore). Nuclei were visualized by
co-staining with DAPI and data was imaged with an Olympus BX63.
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