Cells from one well of 6-well dishes were lysed in 80μ1 of lysis buffer (10 mM Hepes pH7.5, 100 mM NaCl, 1 mM EDTA, and 1% NP40), and l/8th of the cell lysate was subjected to Western blot analysis. Proteins were separated by electrophoresis in SDS-12% polyacrylamide gel. Following transfer, the blot was incubated at 4°C overnight with a 1:4000 dilution of rabbit polyclonal anti-HBs antibody (Novus) (Zhang et al., 2017 (link)). After further incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody at 1:10,000 dilution, signals were revealed by enhanced chemiluminescence (PerkinElmer) and visualized by chemiluminescent imaging system (Tanon). The antibodies were removed by the stripping buffer (CWBio), and the blot was incubated sequentially with mouse anti-actin antibody (Proteintech) (1:3000 dilution) and HRP-conjugated goat antimouse antibody (1:10,000 dilution).
Mouse anti actin antibody
Manufactured by Proteintech
Sourced in United States, China
The Mouse anti-actin antibody is a primary antibody that recognizes actin, a highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody can be used to detect and quantify actin in various experimental applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Stripping buffer, by CWBIO (1 mentions)
Enhanced chemiluminescence, by PerkinElmer (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Bio-Rad (1 mentions)
Fluorescence microscopy, by Zeiss (1 mentions)
Rabbit anti flag antibody, by Proteintech (1 mentions)
Goat anti mouse igg h l alexa fluor plus 555, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Sds page, by Beyotime (1 mentions)
Cdyssey clx, by GE Healthcare (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
4 protocols using mouse anti actin antibody
1
Western Blot Analysis of HBs Protein
2
Silencing ADORA1 and GAPDH in Cells
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Small interfering RNAs (siRNAs) to ADORA1, GAPDH, and Negative Control were purchased from Ambion (Austin, TX, U.S.A.). Lipofectamin (Lipofectamine RNAiMAX) was purchased from Invitrogen (Carlsbad, CA, U.S.A.). Tween 20 and bovine serum albumin-fraction V (BSA) were purchased from RPI (Mount Prospect, IL, U.S.A.). Rabbit anti-ADORA1 antibody was purchased from Sigma-Aldrich (Saint Louis, MO, U.S.A.). Mouse anti-actin antibody was purchased from Proteintech (Rosemont, IL, U.S.A.). Mouse anti-caspase 3 and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Goat anti-rabbit Poly-HRP and goat anti-mouse IgG (H+L) Poly-HRP secondary antibodies were purchased from Invitrogen. Pre-casted 4–20% gradient gels, polyvinylidene fluoride (PVDF) membrane and other laboratory reagents were purchased from Bio-Rad (Hercules, CA, U.S.A.).
3
Immunofluorescence Localization of Actin and Flag
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Forty-eight hours after transfection, cells were seeded on glass coverslips and then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked for 1 h in 2% BSA. Immunostaining was conducted with mouse anti-actin antibody (1 : 1000, ProteinTech) and rabbit anti-Flag antibody (1 : 200, ProteinTech) overnight at 4°C. Goat anti-mouse IgG (H+L) Alexa Fluor Plus 555 and goat anti-rabbit IgG (H+L) Alexa Fluor Plus 488 secondary antibodies (Invitrogen, USA) were used at 1 : 1000 at room temperature for 1 h. The cell nuclei were stained with DAPI (6-diamidino-2-phenylindole). Protein localization was observed by fluorescence microscopy (Carl Zeiss, Germany).
4
Western Blot Analysis of BRMS1 Protein
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Transfected cells were harvested and washed twice with PBS (Solarbio, Beijing, P.R. China). Cell lysis buffer (120 μl; Thermo Fisher) was added to each cell and incubated on ice for 30 min. After centrifugation at 4°C, 13,000 rpm, for 10 min, the supernatant was carefully collected to obtain the total protein. Protein concentration was measured using the BCA protein assay kit (Solarbio). Equal amounts of protein (30 μg) were separated by 12% SDS-PAGE (Beyotime) and then transferred to PVDF membranes (Thermo Fisher). Membranes were blocked with Tris-buffered saline (TBS; Solarbio) containing 5% fat-free milk powder (Yili, Beijing, P.R. China) at room temperature for 1 h. Then the membrane was incubated overnight at 4°C with rabbit anti-BRMS1 antibody (Proteintech, Wuhan, Hubei, P.R. China) or mouse anti-actin antibody (Proteintech). The following day, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase against mouse IgG or rabbit IgG (Proteintech) for 1 h at room temperature after washing three times with TBST. Blots were detected with the infrared laser scanning imaging system (CDYSSEY CLx; General Electric Company).
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