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).
Catalog no mms 118p
Manufactured by Merck Group
The Merck Catalog No. MMS-118P is a laboratory equipment item. It serves a core function within the scientific research and testing processes. Detailed specifications and intended use are not available in this factual, unbiased description.
Automatically generated - may contain errors
2 protocols using catalog no mms 118p
1
Western Blot Analysis of Transfected COS1 Cells
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).
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