Proteins were extracted using radioimmunoprecipitation assay buffer (P0013B; Beyotime, Shanghai, China). The protein samples were first separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Immobilon, Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBST buffer (TBS containing 0.1% Tween-20) at room temperature for 1 h, and then incubated with one of the following primary antibodies at 4°C overnight: rabbit anti-BAX (1:5000; Proteintech), rabbit anti-Bak (1:1000; Proteintech), rabbit anti-Bcl-2 (1:2000; Proteintech), and rabbit anti-β-actin (1:1000; Proteintech). After washing, the membranes were incubated with the appropriate secondary antibody (anti-rabbit Ig-G, 1:2000; Proteintech) for 1 h. The immunoreactive proteins were quantified using the NIH ImageJ software. β-Actin was used as an internal control. The protein levels are expressed as protein/β-actin ratios.
Radioimmunoprecipitation assay buffer
Manufactured by Beyotime
Sourced in China, United States, Switzerland, Germany
Radioimmunoprecipitation assay buffer is a solution used in molecular biology techniques to extract and solubilize proteins from cells or tissues. It is a key component in the radioimmunoprecipitation assay (RIPA) procedure.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (47 mentions)
Bca protein assay kit, by Beyotime (42 mentions)
Polyvinylidene difluoride membrane, by Merck Group (36 mentions)
Pvdf membrane, by Merck Group (28 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (21 mentions)
Bicinchoninic acid assay, by Beyotime (17 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (14 mentions)
Gapdh, by Abcam (13 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Quantity one software, by Bio-Rad (11 mentions)
Nitrocellulose membrane, by Merck Group (10 mentions)
Ab9485, by Abcam (9 mentions)
Ab205718, by Abcam (9 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (9 mentions)
Image lab software, by Bio-Rad (9 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
Bicinchoninic acid kit, by Beyotime (9 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (9 mentions)
Enhanced chemiluminescence reagent, by Merck Group (9 mentions)
455 protocols using radioimmunoprecipitation assay buffer
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of EMT Markers
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Cells were lysed by Radio Immunoprecipitation Assay (RIPA) buffer (Radio-Immunoprecipitation assay buffer, Beyotime). Then, bicinchoninic acid (BCA) Protein Assay Kit (Beyotime) was applied to detect the concentrations of above protein samples. Afterwards, proteins were segregated with the 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA, USA). After blocking, membranes were incubated with primary antibodies against FOXD1 (ab49156; 1:1000 dilution), E-cadherin (ab40772; 1:10,000 dilution), N-cadherin (ab18203; 1:1000 dilution), Vimentin (ab45939; 1:1000 dilution), ERK1/2 (ab17942; 1:1000 dilution), phosphorylated ERK1/2 (ab223500; 1:1000 dilution) and GADPH (ab8245; 1:10,000 dilution) at 4 °C overnight. All antibodies were provided by Abcam Company (Cambridge, UK). Subsequently, the membranes were cultured with the corresponding secondary antibodies, followed by analysis of the bands by the enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology, Rockford, IL, USA).
3
Western Blot Analysis of Protein Expression
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Cells and human Bca tissues were lysed in radioimmunoprecipitation assay buffer (Beyotime, Beijing, People’s Republic of China), supplemented with protease inhibitors (Roche, Shanghai, People’s Republic of China) and the serine protease inhibitor phenylmethylsulfonyl fluoride (Roche), at 4°C for 1 h. Proteins from tissues and cells were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and were then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated first with primary antibodies, then with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Blots were detected using a Bio-Rad Bioimaging system (Bio-Rad, Hercules, CA, USA), and antibodies against GAPDH served as a negative control. Rabbit monoclonal antibodies (1:1,000) against TGF-β, Smad2/3, phosphorylated Smad2 (p-Smad2), p-Smad, vimentin, N-cadherin, and matrix metallopeptidase-9 (MMP-9) were purchased from Cell Signaling Technology (St Louis, MO, USA). Rabbit monoclonal antibody against Trim59 (1:200) was purchased from Abcam (Cambridge, MA, USA). All experiments were performed in triplicate.
4
Protein Extraction and Western Blotting from BM-MSCs
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Proteins were extracted from the BM-MSCs using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Beijing, China) and the total protein concentration was measured using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Proteins (40–60 μg/lane) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA) for 1 h at 100 mA. Membranes were incubated in 3% bovine serum albumin/PBS-Tween for 1 h at room temperature. Primary antibodies were used at a dilution of 1:1,000 and applied by overnight (at least 12 h) incubation at 4°C. Subsequently, the membrane was incubated with a fluorescent-conjugated secondary antibody at a dilution of 1:10,000 for 1 h. Blots were analyzed using the Odyssey imaging system (LI-COR Biosciences).
5
Quantifying ΔNP63 Protein Levels
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The tissues and cells lysates were prepared with radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China). The supernatant of lysates was collected after centrifugation, and the protein concentration was determined with the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. The Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad). The △NP63 protein level was analyzed by western blot with antibody for △NP63 (poly6190, BioLegend, CA, USA). The protein levels were normalized by probing the same blots with a GAPDH antibody (FL-335, sc-25778, Santa Cruz Biotechnology, CA, USA).
6
Western Blot Analysis of Skin Proteins
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Skin tissue samples were lysed in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) and total protein concentration was measured with a bicinchoninic acid assay kit (Beyotime). Western blotting was performed as previously described [22 (link)] using antibodies against the following proteins: MMP-1 (rabbit polyclonal, 1 : 1000, Abcam, Cambridge, UK, cat. number ab137332); TIMP-1 (rabbit polyclonal, 1 : 200, Santa Cruz Biotechnology, Santa Cruz, CA, USA, cat. number 5538); and JNK (rabbit monoclonal, 1 : 1000, cat. number 9252), phospho-JNK (rabbit monoclonal, 1 : 1000, cat. number 4668), p38 mitogen-associated protein kinase (MAPK) (rabbit polyclonal, 1 : 1000, cat. number 9212), and p-p38 (rabbit polyclonal, 1 : 1000, cat. number 9211) (all from Cell Signaling Technology, Danvers, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (mouse monoclonal, 1 : 20,000, Sigma, St. Louis, MO, USA, cat. number G9295) served as a loading control. Protein band intensity was quantified using Gene Tools image analysis software (Syngene, Cambridge, UK).
7
Quantifying Osteoclast Protein Levels
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Western blot analysis was performed to detect protein expression levels. Total protein was isolated from mature osteoclasts using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Equal amounts of protein (25 µg/lane) were separated by SDS-PAGE on 10% gels and transferred onto PVDF membranes. After blocking with 5% non-fat skim milk at room temperature for 2 h, the membranes were incubated at 4°C overnight with primary antibodies against P2X7 (cat. no. NBP1-20180; 1:1,000; Novus Biologicals, LLC), LC3 (cat. no. L7543; 1:1,000; MilliporeSigma), TRAP (cat. no. ab238033; 1:1,000), CK (cat. no. ab37259; 1:1,000), CAII (cat. no. ab182611; 1:1,000), MMP-9 (cat. no. ab76003; 1:1,000) (all from Abcam), NFATc1 (cat. no. 8032; 1:1,000), c-fos (cat. no. 2250; 1:1,000), calcineurin (cat. no. 2614; 1:1,000), Beclin-1 (cat. no. 3495; 1:1,000) and β-actin (cat. no 4970; 1:1,000; all from Cell Signaling Technology, Inc.). Subsequently, membranes were incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (cat. nos. 7074 and 7076; 1:1,000; Cell Signaling Technology, Inc.). The proteins were visualized using an ECL kit (cat. no. P2300; New Cell & Molecular Biotech) and the data were analyzed using ImageJ software; the protein expression levels were calculated according to the gray value of the bands.
8
Western Blot Analysis of Apoptosis Markers
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Cultured cells or frozen cardiac muscle tissues were lysed using radioimmunoprecipitation assay buffer (Cat: P0013C; Beyotime) to obtain total protein.39 Then, the proteins were separated on an sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (Cat: P0012AC; Beyotime) and were transferred to a Hybond‐PVDF membrane (Cat: FFP36; Beyotime). Next, membranes were blocked in 5% milk for 2 h at room temperature (RT) and incubated with primary antibodies overnight at 4°C. The primary antibodies contain Creb1 (rabbit, 1:500, ab32515), Bax (rabbit, 1:1000, ab32503), Bcl‐2 (rabbit, 1:500, ab182858), cleaved‐caspase‐3 (rabbit, 1:500, ab32042), and β‐actin (rabbit, 1:500, ab7817). Following three times washes using TBST, the membrane was incubated with specific secondary antibodies (goat anti‐rabbit IgG, 1:10,000, ab6721) for 1 h at RT, and was washed with TBST three times. The ChemiDoc XRS Imaging System (BioRad) was used to detect and quantify the specific protein. Grayscale determination was performed and statistical analysis using Fiji.40
9
Protein Expression Analysis via Western Blot
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Total cell protein was extracted in Radio Immunoprecipitation Assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). The protein extracts were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After blocking, the membrane was probed with primary antibody against a-SMA (1:1000), FN (1:1000; Protein-Tech, Chicago, IL, USA), and GAPDH (1:2000; Millipore, Billerica, MA, USA), followed by the appropriate horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (Millipore). Specific bands were visualized by a standard enhanced chemiluminescence procedure (Millipore). The signals were analyzed using Image-Pro Plus (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). The band density of each sample was normalized to the GAPDH band.
10
Autophagy Signaling Pathway Evaluation
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Treated cells were washed with cold PBS and lysed with radioimmunoprecipitation assay buffer (Beyotime, China) supplemented with protease inhibitors to extract total protein. Protein concentration was determined by the bicinchoninic acid protein assay. After denaturation, the proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Nonspecific binding was blocked with 5% skimmed milk in TBST buffer for 2 h, followed by incubation with primary antibodies (all 1:1,000; Abcam, Cambridge, UK) at 4°C overnight and secondary antibodies (1:5,000; Beyotime Biotechnology, Shanghai, China) at room temperature for 2 h. The blots were visualized using ECL detection reagents.
The list of antibodies is as follows: LC3II (ab192890), p62 (ab207305), beclin-1 (EPR20473), ROS (ab108492), JNK (ab273417), p53 (ab26), caspase-3 (ab32351), caspase-8 (ab32125), b-actin (ab8227), AMPK (ab32047), p-AMPK (ab92701), mTOR(ab134903), p-mTOR(ab109268), ULK1(ab240916), and p-ULK1(ab133747).
The list of antibodies is as follows: LC3II (ab192890), p62 (ab207305), beclin-1 (EPR20473), ROS (ab108492), JNK (ab273417), p53 (ab26), caspase-3 (ab32351), caspase-8 (ab32125), b-actin (ab8227), AMPK (ab32047), p-AMPK (ab92701), mTOR(ab134903), p-mTOR(ab109268), ULK1(ab240916), and p-ULK1(ab133747).
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