Cell protein lysates were separated by 10% SDS-PAGE and blotted onto a polyvinylidene fluoride (PVDF) membrane (Roche Diagnostics, Mannheim, Germany). After soaking in 10 ml of 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) solution for 1 h, the membrane was incubated with human polyclonal NOTCH3 (1:500 dilution; Abcam, Cambridge, UK), monoclonal NICD3 (1:1,000 dilution; Abcam), monoclonal HES1 (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), and polyclonal antibody against GAPDH (1:2,000 dilution; Sigma-Aldrich, St. Louis, MO, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody (1:5,000 dilution; Sigma-Aldrich). Results were visualized following treatment with enhanced chemiluminescent (ECL) substrate.
Polyclonal antibody against gapdh
Manufactured by Merck Group
Sourced in United States
Polyclonal antibody against GAPDH is a laboratory reagent used in various research and diagnostic applications. It is a mixture of antibodies that recognize multiple epitopes on the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a commonly used reference or housekeeping gene in molecular biology and cell biology studies.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated goat anti rabbit igg, by Merck Group (2 mentions)
Pvdf membrane, by Roche (1 mentions)
Notch3, by Abcam (1 mentions)
P27kip1, by Cell Signaling Technology (1 mentions)
Ecl reagent kit, by Merck Group (1 mentions)
Scion image software, by Techcomp Instruments (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
2 protocols using polyclonal antibody against gapdh
1
Western Blot Analysis of Notch Signaling
2
Western Blot Analysis of Notch3, Hes1, and p27Kip1
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The cultured cells were washed twice with ice-cold PBS and lysed on ice with RIPA lysis buffer containing freshly added protease and phosphatase inhibitor cocktails. After 15 min of incubation, cell lysate was centrifuged for 10 min (15,000 × g) at 4 °C; supernatant was saved as total protein. Equivalent amounts of protein (30 μg) from each sample were separated on SDS–polyacrylamide gels, then transferred to nitrocellulose membranes (Bio-Rad). Monoclonal antibodies against Notch3 (Abcam, 1:500 dilution) and Hes1 (Cell Signaling Technology, 1:1000 dilution), p27Kip1 (Cell Signaling Technology, 1:1000 dilution) as well as polyclonal antibody against GAPDH (Sigma–Aldrich, 1:2000 dilution) were used according to the manufacturer instructions. Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody (Sigma–Aldrich, 1:5000 dilution). Blots were developed using ECL reagent kit (Millipore). The signal intensity of the appropriate bands on the autoradiogram was calculated by using Scion Image software (Scion).
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