The protein used for Western blotting was extracted using RIA lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) supplemented with protease inhibitors (Roche, Basel, Switzerland). The proteins were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The Western blot system was established using a Bio-Rad Bis-Tris Gel system according to the manufacturer’s instructions. Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1,000. The primary antibody was incubated with the membrane at 4°C overnight, followed by washing and incubation with the secondary antibody marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride (PVDF) membrane carried blots, and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS system, and then 200 μl of Immobilon Western Chemiluminescent HRP Substrate (Millipore, Boston, MA, USA) was added to cover the membrane surface. The signals were captured, and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, P.R. China).
Ria lysis buffer
Manufactured by Beyotime
Sourced in China
The RIA lysis buffer is a laboratory reagent designed to facilitate the extraction and purification of proteins from biological samples. It is a key component in radioimmunoassay (RIA) procedures, which are used to quantify the concentration of specific proteins or other analytes in a sample. The buffer's function is to efficiently break down cell membranes and release the target proteins, allowing for their subsequent analysis and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (4 mentions)
Bis tris gel system, by Bio-Rad (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Chemidoc xrs system, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Merck Group (1 mentions)
4 protocols using ria lysis buffer
1
Western Blot Protocol with Chemiluminescent Detection
2
Western Blot Analysis of Protein Extracts
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The protein used for western blotting was extracted using RIA lysis buffer (Beyotime Biotechnology, China) supplemented with protease inhibitors (Roche, China). The proteins were quantified using the BCA™ Protein Assay Kit (Pierce, USA). Nuclear extracts and cytoplasmic extracts were prepared as previously described (16 (link)). The western blot system was established using a Bio-Rad Bis-Tris Gel system according to the manufacturer's instructions. Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1,000. Primary antibody was incubated with the membrane at 4°C overnight, followed by washing and incubation with secondary antibody marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride (PVDF) membrane with blots and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS system, and then 200 µL Immobilon Western Chemiluminescent HRP Substrate (USA) was added to cover the membrane surface. The signals were captured and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, China).
3
Western Blot Analysis of Signaling Pathways
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Cellular protein was extracted using the RIA lysis buffer (Beyotime
Biotechnology, Shanghai, China). The purity of the extracts was tested by BCA™
Protein Assay Kit (Pierce, Appleton, WI, USA). Proteins were separated by the
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were
transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA,
USA). After blocking with 5% non-fat milk for 1 h, the membranes were probed by
the antibodies at 4°C overnight, for the detection of Bcl-2 (ab692), Bax
(ab77566), pro-caspase-3 (ab4051), cleaved-caspase-3 (ab13847), IL-6 (ab6672),
TNF-α (ab6671), PI3K (ab191606), p-PI3K (ab182651), AKT (ab8805), p-AKT
(ab38449), IκBα (ab32518), p-IκBα (ab133462), p65 (ab16502), p-p65 (ab86299),
Collagen II (ab188570), Aggrecan (ab3778), MMP-3 (ab53015), MMP-13 (ab51072),
and β-actin (ab8226, Abcam, Cambridge, MA, USA). The membranes were then
incubated with the secondary antibodies for 1 h at room temperature. Signals
were developed using ECL Plus Western Blotting Substrate (Pierce, Carlsbad, CA,
USA). The intensity of the bands was quantified using Image Lab™ Software
(Bio-Rad, Shanghai, China).
Biotechnology, Shanghai, China). The purity of the extracts was tested by BCA™
Protein Assay Kit (Pierce, Appleton, WI, USA). Proteins were separated by the
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were
transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA,
USA). After blocking with 5% non-fat milk for 1 h, the membranes were probed by
the antibodies at 4°C overnight, for the detection of Bcl-2 (ab692), Bax
(ab77566), pro-caspase-3 (ab4051), cleaved-caspase-3 (ab13847), IL-6 (ab6672),
TNF-α (ab6671), PI3K (ab191606), p-PI3K (ab182651), AKT (ab8805), p-AKT
(ab38449), IκBα (ab32518), p-IκBα (ab133462), p65 (ab16502), p-p65 (ab86299),
Collagen II (ab188570), Aggrecan (ab3778), MMP-3 (ab53015), MMP-13 (ab51072),
and β-actin (ab8226, Abcam, Cambridge, MA, USA). The membranes were then
incubated with the secondary antibodies for 1 h at room temperature. Signals
were developed using ECL Plus Western Blotting Substrate (Pierce, Carlsbad, CA,
USA). The intensity of the bands was quantified using Image Lab™ Software
(Bio-Rad, Shanghai, China).
4
Western Blot Protein Extraction and Analysis
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The proteins used for western blotting assay were extracted by using radioimmunoassay (RIA) lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Guangzhou, China). The protein samples were quantified by the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Western blot system was established by using a Bio-Rad Bis-Tris Gel system according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Sigma. Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1000, then been incubated with the polyvinylidene difluoride (PVDF) membranes at 4 °C overnight, followed by wash and incubation with secondary antibody marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the membranes carried blots and antibodies were transferred into Bio-Rad ChemiDoc™ XRS system, and then 200 μl Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) was added to cover the membrane surface. The signals were captured and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, China).
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