The protein concentrations of cell and EV lysates were determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA). The lysates were normalized by protein concentration, resolved by SDS-PAGE, and then the proteins were transferred to PVDF membranes. The membranes were incubated with various primary antibodies including β-actin (Catalog No. ab8226; Abcam, Cambridge, MA, USA), Survivin (Catalog No. NB500-201; Novus Biologicals), flotillin-2 (Catalog No. 3436S; Cell Signaling), CD-63 (Catalog No. 10628D; ThermoFisher, Waltham, MA, USA), and IκBα (Catalog No. 9242; Cell Signaling), diluted in in 20 mM Tris, 135 mM NaCl, and 0.02% Tween 20 (TBST). The primary antibodies were detected with HRP-conjugated secondary antibodies (Catalog Nos. 7074S and 7076S; Cell Signaling) followed by exposure to ECL (enhanced chemiluminescence) reagent (Catalog No. 32106; ThermoFisher).
Catalog no 32106
Manufactured by Thermo Fisher Scientific
The Catalog No. 32106 is a laboratory equipment product. It is a compact and portable device designed for performing various analytical tasks in the laboratory environment. The core function of this product is to provide reliable and precise measurements for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Catalog no a7906, by Merck Group (2 mentions)
Lab catalog no 580 2, by Fortrea (2 mentions)
Catalog no mms 118p, by Merck Group (2 mentions)
Catalog no cp06, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Flotillin 2, by Cell Signaling Technology (1 mentions)
3 protocols using catalog no 32106
1
Quantifying Cellular and Extracellular Vesicle Proteins
2
Western Blot Analysis of Transfected COS1 Cells
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Transfected COS1 cells were washed with chilled phosphate buffered saline solution and lysed in RIPA buffer at 4 °C. Extracts were precleared by centrifugation at 13 200 rpm for 10 min at 4 °C, denatured by boiling in SDS-PAGE sample buffer, separated electrophoretically, and transferred onto nitrocellulose filters using the iBlot Dry Blotting System (Thermo Fisher Scientific, Catalog No. IB21001). Membranes were blocked in 5% bovine serum albumin (Sigma-Aldrich, Catalog No. A7906) in TBS-T for at least 1 hour and then incubated overnight at 4 °C with the appropriate antibodies to Vav2 (home-made; Lab catalog No. 580-2, 1:1000 dilution), EGFP (1:5000 dilution, Covance, Catalog No. MMS-118P), and tubulin α (1:2000 dilution, Calbiochem-Merck, Catalog No. CP06). After three washes with TBS-T to eliminate the primary antibody, the membranes were incubated with the appropriate secondary antibody (1:5000 dilution, GE Healthcare) for 30 min at room temperature. Immunoreacting bands were developed using a standard chemiluminescent method (Thermo Fisher Scientific, Catalog No. 32106).
3
Western Blot Analysis of Transfected COS1 Cells
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