To extract protein, culture cells were resuspended in lysis buffer containing protease and phosphatase inhibitors. The samples were centrifuged at 13,000 g for 10 min at 4°C, and the supernatant was collected to quantify the protein by Bradford assay. Protein (15 µg) from each sample was resolved on 12.5% SDS-PAGE gels and transferred to nitrocellulose membranes using the Trans-Blot semi-dry transfer cell system (Bio-Rad). For western blot detection, the membranes were incubated with Intercept (PBS) Blocking Buffer (LI-COR Bioscience) for 1 hr at room temperature with gentle shaking and incubated overnight at 4°C with the primary antibodies against OCT4 and NANOG using GAPDH as internal control (List of antibodies in Supplementary file 5 ). Membranes were rinsed with 1× PBS with 0.1% Tween 20 to incubate with the LI-COR secondary antibodies (List of antibodies) for 1 hr at room temperature. Scanning of the membranes was carried out with an Odyssey CLx System (LI-COR Bioscience), and the fluorescence intensity was analyzed with Image Studio version 4.0 software (LI-COR Bioscience).
Trans blot semi dry transfer cell system
Manufactured by Bio-Rad
The Trans-Blot semi-dry transfer cell system is a laboratory equipment used for the electrophoretic transfer of proteins and nucleic acids from a gel to a membrane or other solid support. The system utilizes a semi-dry transfer method to efficiently and consistently transfer biomolecules from the gel to the desired membrane.
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2 protocols using trans blot semi dry transfer cell system
1
Quantitative Protein Analysis by Western Blot
2
Detecting GalNAc-Binding Proteins by Blotting
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Electrophoretically separated SPP and SAP samples were immobilized on a 0.45-μm nitrocellulose membrane (Sigma) using a Trans-Blot Semi-Dry Transfer Cell system (Bio-Rad). GalNAc-binding proteins were detected according to the methods of Ignotz et al. ( 2001), with modifications. Briefly, membranes were incubated for 1 h at room temperature in binding buffer (HBSS with 5% bovine serum albumin, 1 mM NiCl2, and 0.05% [v/v] Tween20) to block any non-specific sites and to renature the blotted proteins. After three rinses in wash buffer (HBSS with 1 mM NiCl2, 0.5% [v/v] Tween20), membranes were probed with 5 µg/ml GalNAc-polyacrylamide-FITC in binding buffer for 2 h at room temperature, followed by three 10-min washes.
Membranes were then incubated with alkaline phosphatase-conjugated mouse monoclonal anti-FITC (Sigma) (1:10000 in binding buffer) for 1 h at room temperature, followed by additional washes. Visualization of GalNAc-binding proteins was achieved by incubating in 23.25 mg/ml SigmaFast chromogenic substrate (BCIP/NBT) (Sigma) until a violet precipitate developed.
Membranes were then incubated with alkaline phosphatase-conjugated mouse monoclonal anti-FITC (Sigma) (1:10000 in binding buffer) for 1 h at room temperature, followed by additional washes. Visualization of GalNAc-binding proteins was achieved by incubating in 23.25 mg/ml SigmaFast chromogenic substrate (BCIP/NBT) (Sigma) until a violet precipitate developed.
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