The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) using a Bio-Rad semidry blotter (Bio-Rad, USA). The membranes were probed using the method previously described (55 (link)). In short, the membrane was blocked in 20 mM Tris-buffered saline with 0.05% Tween 20 (TBST) (pH 8.0) in 5% skim milk for 1 h at room temperature and then incubated with the primary antibody for 1 h or for 12 h at 4°C. Subsequently, the treated membrane was washed three times with TBST for 15 min each before incubating with a secondary antibody for 1 h at room temperature. After three more washes in TBST for 15 min each, the immune-reactive proteins could be localized. The membranes were treated with Clarity Western ECL substrate (number 170-5061; Bio-Rad, USA) for 5 min under dark conditions. The primary antibody used is monoclonal mouse anti-FLAG (number M20008S; Abmart, Shanghai, China) at a dilution of 1:5,000. The secondary antibody used is goat anti-mouse secondary antibody (number M21001L; Abmart, Shanghai, China) at a dilution of 1:5,000. Immune-reactive bands were visualized by the VIBER molecular imaging system (Fusion FX7, France).
Goat anti mouse secondary antibody
Manufactured by Abmart
Sourced in China
Goat anti-mouse secondary antibody is a laboratory reagent used to detect the presence of mouse primary antibodies in various immunoassays, such as ELISA, Western blotting, and immunohistochemistry. This secondary antibody is conjugated with a reporter molecule, such as an enzyme or fluorescent dye, to enable visualization and quantification of the target mouse primary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Dimethyl sulfoxide (dmso), by Maclin Biochemical Technology (1 mentions)
Rabbit anti human bcl 2, by Abcam (1 mentions)
Rabbit anti human bax, by Abcam (1 mentions)
Crystal violet solution, by Solarbio (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Mtt assay kit, by Solarbio (1 mentions)
2 protocols using goat anti mouse secondary antibody
1
Western Blot Analysis of Protein Samples
2
Cervical Cancer Cell Line Cultivation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Human cervical cancer cell lines, SiHa (CSTR:19375.09.3101HUMTCHu113) and HeLa (CSTR:19375.09.3101HUMTCHu187), were obtained from Chinese Academy of Sciences Cell Bank (Shanghai, China). Both cells were cultured in Dulbecco's Modi ed Eagle Medium (DMEM; Gbico, NY, USA) containing 4.5 g/L glucose, 10% fetal bovine serum (FBS; Biological Industries, Israel) and 1% penicillin-streptomycin (Sigma-Aldrich, MS, USA). Cells were incubated at 37°C, 5% CO 2 atmosphere and 95% relative humidity. Cells were washed with PBS and substituted with indicated culture medium every 2-3 days. 0.25% trypsin containing EDTA was applied to passage expansion. The major reagents and antibodies used in this study included dimethyl sulfoxide and PTS (Macklin, Shanghai, China) as well as the following antibodies: rabbit anti-human MICA/B, rabbit anti-human p53, rabbit anti-human bcl-2 and rabbit anti-human Bax (all obtained from Abcam, UK), rabbit anti-human PI3K (110α), rabbit anti-human phosphorylated PI3K (p-PI3K) p55 (Tyr199), rabbit anti-human AKT, rabbit anti-human p-AKT (Ser473), goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody (all obtained from Abmart, Shanghai, China). SAHA (molecular weight, 264.32) was purchased from Sigma (No. 149647-78-9). 0.5% crystal violet solution and MTT assay kit were obtained from Solarbio Life Sciences (Beijing, China).
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