Frozen tumor and breast tissue were weighed, mechanically dissociated and lysed in M-PER solution followed by protein quantification using standard Bradford assay. 40 μg protein was electrophoresed on a 15% SDS-polyacrylamide gel (Bio-Rad Hercules, CA), transferred to a PVDF membrane (Millipore, Billerica, MA), blocked in 3% Bovine Serum Albumin (Sigma Aldrich, St. Louis, MO, Catalog #A7030) overnight and probed with rabbit anti-mouse primary antibodies against α-Lactalbumin (U.S. Biologicals, Salem, MA, Catalog #037664). Mouse anti-human β-Actin primary antibody (Proteintech, Rosemont, IL, Catalog #60008-1) was probed on the same blot as loading and protein expression control. Primary antibody binding was detected using HRP conjugated goat anti-rabbit (Santa Cruz Biotechnologies, Dallas, TX) or anti-mouse secondary antibodies (Promega) followed by detection of chemiluminescence on X-Ray films (Denville Scientific, Holliston, MA). Intensity of specific protein bands was quantified using the Image Studio software (LI-COR, Lincoln, Nebraska) and normalized to intensity of β-Actin control bands.
Anti mouse secondary antibodies
Manufactured by Promega
Anti-mouse secondary antibodies are laboratory reagents used to detect and visualize mouse primary antibodies in various immunological techniques, such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
X ray film, by Thomas Scientific (1 mentions)
Hrp conjugated goat anti rabbit, by Santa Cruz Biotechnology (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Image studio software, by LI COR (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bcl 2, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Thermo Fisher Scientific (1 mentions)
Anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
2 protocols using anti mouse secondary antibodies
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of Apoptosis Regulators
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Cells were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer. After centrifugation, the supernatant was quantified with the bicinchoninic acid colorimetric (BCA) method. Next, 30 µg of protein was separated on a 10% SDS-PAGE gel and transferred to PVDF membranes in a pre-assembled transfer sandwich process. After blocking with 5% non-fat blocking grade milk at room temperature for 1 h, the membranes were blotted overnight at 4°C with antibodies against Bax (cat#: PA5-39778, 1:1,000, Thermo Fisher Scientific), Bcl-2 (cat#: 13-8800, 1:1,000, Thermo Fisher Scientific), PTPRB (cat#: PA5-119373, 1:1,000, Thermo Fisher Scientific). Anti-rabbit secondary antibody (cat#: sc-3836, 1:5,000, Santa Cruz Biotechnology) and anti-mouse secondary antibodies (cat#: W4021, 1:10,000; Promega) were used to identify the target proteins on the membranes 24 h later. ECL Plus reagents were used to detect the immunoreactive bands (Amersham).
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