Cells were lysed in RIPA Buffer (Cell Signaling Technology, 9800), and protein concentrations were determined by using BCA assays (Cell Signaling Technology, 7780). Total protein (40 mg) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% gel and electro transferred to polyvinylidene fluoride membranes. Membranes were then blocked with 5% nonfat dry milk in Tris-buffered saline (Sigma, T7777, pH 7.6) and were incubated overnight at 4 ℃ with rabbit monoclonal anti-N-cadherin, anti-LC3B, anti-Beclin-1, anti-ATG7 antibodies (Cell Signaling Technology, EMT Antibody Sampler Kit 9782 and Autophagy Antibody Sampler Kit 4445) and rabbit polyclonal antibody anti-T-cadherin (Millipore, ABT121). Alternatively, membranes were incubated with mouse monoclonal anti-GAPDH (Abclonal, AC002), apoptosis antibody sampler kit (Cell Signaling Technology 9915), anti-phospho SAPK/JNK (Cell Signaling Technology 81E11) and anti-SAPK/JNK (Cell Signaling Technology 9252), anti-Akt (Cell Signaling Technology C67E7) and anti-phospho Akt antibodies (Cell Signaling Technology D9E). HRP-conjugated anti-rabbit IgG and anti-mouse IgG antibodies were obtained from Cell Signaling Technology (7074).
Bca assay
Manufactured by Cell Signaling Technology
Sourced in United States
BCA assays are a colorimetric detection method used to quantify the total protein concentration in a sample. The assay relies on the reduction of copper ions by protein in an alkaline medium, resulting in a purple-colored reaction that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Cell Signaling Technology (4 mentions)
Horseradish peroxidase linked secondary antibody, by Jackson ImmunoResearch (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Enhanced chemiluminescence system, by GE Healthcare (3 mentions)
Anti phospho sapk jnk, by Cell Signaling Technology (1 mentions)
Anti sapk jnk, by Cell Signaling Technology (1 mentions)
Anti phospho akt antibody, by Cell Signaling Technology (1 mentions)
Tris buffered saline (tbs), by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit igg and anti mouse igg antibodies, by Cell Signaling Technology (1 mentions)
Apoptosis antibody sampler kit, by Cell Signaling Technology (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (1 mentions)
Las 4000 image document instrument, by Fujifilm (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ecl western blotting substrate, by Promega (1 mentions)
Ab37168, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
7 protocols using bca assay
1
Western Blot Analysis of EMT and Autophagy Markers
2
Western Blotting for Protein Analysis
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Western blot analyses were described in a previous study [23 (link)]. Briefly, cells were harvested by low-speed centrifugation and washed with PBS. Cells were lysed in RIPA Buffer (Cell Signaling Technology, 9800), and protein concentrations were determined using BCA assays (Cell Signaling Technology, 7780). Forty micrograms of each protein sample were separated using 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 1 × Tris-buffered saline with Tween 20 (TBST) and 5% (w/v) non-fat milk for 1 h at room temperature. Then, membranes were incubated with primary antibodies overnight at 4 °C. Subsequently, blots were incubated with horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch) for an additional 1 h. Immunoreactive bands were visualized using an enhanced chemiluminescence system (GE Healthcare).
3
Western Blot Protocol for Protein Analysis
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Western blot analyses were performed as previously described (19 (link)). Briefly, cells were harvested by low-speed centrifugation and washed with PBS. Cells were lysed in radioimmunoprecipitation assay buffer (Cell Signaling Technology, Boston, MA, USA), and protein concentrations were determined using bicinchoninic acid (BCA) assays (Cell Signaling Technology). Forty micrograms of each protein sample was separated using 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 1× tris-buffered saline with Tween 20 and 5% (w/v) nonfat milk for 1 hour at room temperature. Then, membranes were incubated with primary antibodies overnight at 4°C. Subsequently, blots were incubated with horseradish peroxidase–linked secondary antibodies (Jackson ImmunoResearch) for an additional 1 hour. Immunoreactive bands were visualized using an enhanced chemiluminescence system (GE Healthcare).
4
Western Blot Protein Analysis Protocol
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Western blot analyses were performed as previously described49 (link). In brief, cells were harvested by low-speed centrifugation and washed with PBS. Cells were then lysed in cold RIPA Buffer (Cell Signaling Technology, Boston, MA, USA), and protein concentrations were measured using BCA assays (Cell Signaling Technology, Boston, MA, USA). Protein samples (40 μg each) were separated on 12% SDS polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes (Bio-Rad). The membranes were incubated for 1 h at room temperature with 1× Tris-buffered saline with Tween 20 and 5% (w/v) non-fat milk to block non-specific binding. Following the membrane incubation at 4 °C overnight with target-specific primary antibodies, blots were incubated with horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch) for an additional 1 h and the result was normalized to reference internal control. The signal of immunoreactive bands was visualized using a LAS-4000 image document instrument (FujiFilm, Tokyo, Japan).
5
Western Blot Analysis of Protein Expression
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Western blot analyses were performed as described36 (link). Briefly, cells were harvested and lysed in radio immunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, 9800). Protein concentrations were quantified using BCA assays (Cell Signaling Technology, 7780). Cell lysates (40 μg) were electrophoresed using 12% SDS-PAGE, and were then transferred to a nitrocellulose membranes (Bio-Rad) that were subsequently blocked with 5% (w/v) nonfat dried milk. One hour later, blots were incubated with primary antibodies, as indicated in the figures, overnight at 4 °C. Subsequently, immunocomplexes were incubated with horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch) and then blots were developed using an enhanced chemiluminescence system (GE Healthcare).
6
Protein extraction and Western blot
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Cultured cells were pelleted, washed with PBS, and resuspended in RIPA lysis buffer (Cell Signaling, 9806) with 1x protease inhibitor cocktail (MilliporeSigma, P8340). Samples were normalized by bicinchoninic acid (BCA) assay (Cell Signaling, 7780), and loaded on a precast SDS-PAGE gel (Bio-Rad, 4561086). Western blotting followed using standard protocols. Imaging of blots was performed on a LI-COR Odyssey instrument.
7
Western Blot Analysis of CDK6 Expression
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SiHa cells were harvested at 24 h post-transfection and cells were counted. Cell pellets containing 105 SiHa cells were resuspended in 1 mL RIPA solution (Cell Signaling Technology) to extract total proteins. Concentrations of total proteins were measured using BCA assay (Cell Signaling Technology). In a 95°C incubator, protein samples were denatured for 10 min, followed by electrophoresis using 12% SDS-PAGE gel. After that, proteins were transfected to PVDF membranes and blocking was performed in 5% non-fat milk (PBS) at room temperature for 1 h. GAPDH (1:1800, ab37168, Abcam) and CDK6 (1:1800, ab151247, Abcam) rabbit polyclonal primary antibodies were used to incubate with the membranes at 4°C for 18 h, followed by incubation with HRP (IgG) (1:1800; ab6721; Abcam) goat anti-rabbit secondary antibody at 24°C for 2 h. ECL Western Blotting Substrate (Promega Corporation) was dropped onto the membranes and MYECL™ Imager (Bio-Rad) was used to detect signals. Image J v1.46 software was used for data normalizations.
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