The total protein was separated by radioimmunoprecipitation assay lysis buffer (Cw Biotech, Bejing, China) including proteinase suppressors. The protein concentration was assessed by bicinchoninic acid (Beijing ComWin Biotech Co., Ltd., Beijing, China) reagent. After that, 20 µg of protein samples was detected by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, followed by transfer to a polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% fat-free milk for 1 h, the PVDF membranes were hatched with the antibodies at 4°C overnight. The blots were tested with ECL.
Radioimmunoprecipitation assay lysis buffer
Manufactured by CWBIO
Sourced in China
Radioimmunoprecipitation assay lysis buffer is a solution used in the process of protein extraction and purification. Its core function is to disrupt cell membranes and release intracellular proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid kit, by CWBIO (1 mentions)
Mmp13, by Abcam (1 mentions)
Chemiluminescence system, by Bio-Rad (1 mentions)
Col2a1, by Abcam (1 mentions)
P90719, by Merck Group (1 mentions)
Gpadh, by Proteintech (1 mentions)
H3k27me3, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Fluorescent secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ab6992, by Abcam (1 mentions)
Ab124505, by Abcam (1 mentions)
Ab92445, by Abcam (1 mentions)
Ab11734, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab7778, by Abcam (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
9 protocols using radioimmunoprecipitation assay lysis buffer
1
Protein Quantification and Western Blot Analysis
2
Chondrogenic Microsphere Protein Analysis
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The hADSC chondrogenic microspheres and chondrocytes were lysed in radioimmunoprecipitation assay lysis buffer (Cwbio, China). The concentrations of proteins were detected using a bicinchoninic acid kit (Cwbio, China). Protein samples (25 μg) were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, electrophoresis condition: 80 V 25 min, 120 V 60 min; then protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), transfer membrane condition: 250 mA 120 min. The membrane was blocked with 5% skim milk for 60 min. Membranes were incubated with primary antibodies specific for MMP13 (1:1000, Abcam), Col2a1 (1:1000, Abcam), SOX9 (1:1000, Abcam), GPADH (1:000, affinity), KDM6A (1:1000, Proteintech), H3 (1:1000, Proteintech) and H3K27me3 (1:1000, Proteintech). After incubation with a secondary antibody (1:5000, Cell Signaling Technology, Danvers, MA, USA), the signals were detected using a chemiluminescence kit (P90719, Millipore) and chemiluminescence system (Bio‐Rad, Hercules, CA, USA) and analysed using Image Lab Software.
3
Western Blot Analysis of Myocardial Proteins
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Myocardial tissues and H9c2 cells were homogenized in radioimmunoprecipitation assay lysis buffer (CWBIO, Beijing, China), incubated on ice for 20 min, and centrifuged at 14,000 g for 10 min at 4°C. The supernatant was transferred to a tube, and concentrations were detected using the Bradford assay (Bio-Rad Laboratories, CA, USA). Then, 50 μg of protein was transferred to each lane on a 10% SDS-polyacrylamide gel. These samples were transferred to polyvinylidene fluoride membranes (Millipore, MA, USA) and incubated with the following primary antibodies (Supplementary Table S3 ): anti-renin antibody (sc-137252; Santa Cruz Biotechnology, California, USA); anti-angiotensin converting enzyme 1 antibody (ab11734; Abcam, Cambridge, UK); anti-angiotensin II type 1 receptor antibody (ab124505; Abcam, Cambridge, UK); anti-angiotensin II type 2 receptor antibody (ab92445; Abcam); anti-collagen 3 antibody (ab7778; Abcam); anti-CTGF antibody (ab6992; Abcam); and anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH) antibody (ab181602; Abcam) overnight at 4°C. They were incubated with fluorescent secondary antibodies (680 and 790 nm; Jackson, PA, USA) for 2 h during RT. The protein bands were visualized using the Odyssey Imager (Li-Cor Biosciences, NE, USA). The GAPDH protein levels were used as the control protein.
4
Testicular Protein Expression Analysis
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The testicular tissue was lysed on ice with radioimmunoprecipitation assay lysis buffer (Cwbio, Taizhou, China). Subsequently, 20–40 μg of protein was electrophoresed on an 8%–12% (w/v) sodium dodecyl sulfate-polyacrylamide gel gradient and transferred to a nitrocellulose membrane. The membranes were blocked with 5% (w/v) nonfat milk for 1 h in Tris-buffered saline (TBS) and subsequently incubated with the following primary antibodies: rabbit anti-PK2 antibody (1:200, Abcam, Cambridge, MA, USA), rabbit anti-cleaved-caspase-3 antibody (1:1000, Cell Signaling Technology [CST], Boston, MA, USA), rabbit anti-caspase-8 antibody (1:1000, CST), rabbit anti-Bax antibody (1:1000, CST), rabbit anti-Bcl-2 antibody (1:1000, CST), and mouse anti-β-actin antibody (1:500, Boster, Wuhan, China). Immunoreactive bands were detected using electrochemiluminescence (ECL; Pierce, Waltham, MA, USA).
5
Quantitative Western Blot Analysis
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Chondrocytes were washed twice with phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay lysis buffer (CW Biotech, Beijing, China), and the protein concentrations were measured by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc., Waltham, USA). An equal amount of total protein was then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes using β-actin as an endogenous control. The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies, including anti-WISP1 antibody, anti-PI3K antibody, anti-p-PI3K antibody, anti-p65 antibody, anti-p-p65 antibody, anti-β-actin antibody (Abcam, Cambridge, UK). After washing three times with tris buffered saline with Tween-20, the membranes were incubated with the secondary antibody for 1 h at room temperature. Protein bands were visualized using the ECL system, and the optical density of the protein bands was quantified using Image J software.
6
Quantification of Protein Expression by Western Blot
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Western blot was implemented for quantification of protein expression. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and were homogenized with radioimmunoprecipitation assay lysis buffer (Cwbiotech, Bejing, China) containing protease inhibitor. Protein concentration was determined by BCA assay (Cwbiotech, Beijing, China). For Western blot, 40 ng of protein extracts were electrophoresed with 10% sodium dodecyl sulfate polyacrylamide gels, then were transferred to nitrocellulose membrane (Pall Laboratory, NewYork, NY, U.S.A.). The membrane was blocked with 5% non-fat milk at room temperature for 2 h, then incubated with rabbit anti-XRCC4 primary antibody (1:1000, Proteintech, Rosemont, IL, U.S.A.) and anti-GAPDH antibody (1:10,000, Proteintech) at 4°C overnight. The membrane was washed with Tris-buffered saline with 0.1% Tween-20 three times, then was incubated with goat anti-rabbit IgG conjugated horseradish peroxidase secondary antibody (1:5000, Proteintech) at room temperature for 1 h. The signals were detected using Western Lightning Plus ECL (Proteintech) and analyzed by ChemiDoc XRS (Bio-Rad, U.S.A.).
7
Western Blot Analysis of MAPK Signaling
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Total protein was extracted using ice-cold radio-immunoprecipitation assay lysis buffer (Cwbio) containing a phosphatase inhibitor and a protease inhibitor cocktail (both from Roche). Protein from each sample was electro-phoresed in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and then transferred onto a nitrocellulose blot. After 1 hour of blocking with 5% nonfat milk, the blot was incubated overnight at 4°C with antibodies against phospho-JNK (Thr183/Tyr185; 1:1000), and total JNK (1:1000), phospho-p38 MAPK (Thr180/Tyr182; 1:1000), total p38 MAPK (1:1000). On the second day, the blot was washed and then incubated with the respective secondary antibody conjugated to horseradish peroxidase (1:1500) for 1 hour. An enhanced chemiluminescent detection system (Millipore) was used to detect the bands with peroxidase activity. A G-Box iChemi Chemiluminescence image capture system (Syngene) was used to visualize the bands. The same blot was also probed with α-tubulin (1:1000) as internal controls. All antibodies were from Cell Signaling Technology (CST, Massachusetts, USA).
8
Western Blot Analysis of Oxidative Stress Markers
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Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer (CW Biotech) in the presence of a protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). Protein concentrations of cell lysates were quantified using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturers protocol. Total protein (10 ug) were loaded in each well of 12% sodium dodecyl sulfate polyacrylamide gel and subjected to electrophoresis. The proteins were then transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies against GAPDH (1:5,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.), Nrf2 (1:1,000; cat. no. ab62352; Abcam), HO-1 (1:1,000; cat. no. ab13243; Abcam) and NF-κB (1:10,000; cat. no. ab16502; Abcam) at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) for 1 h at room temperature. Detection was performed using ECL Plus western blotting detection reagents (EMD Millipore) and the blots were semi-quantified using ImageJ 2 (National Institute of Health).
9
Western Blot Analysis of Inflammatory Proteins
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Tissue and cell proteins were lysed on ice with radioimmunoprecipitation assay lysis buffer (Cwbio, Taizhou, China). Total proteins were collected after centrifugation. Then, 5 µL of 5× loading buffer was added to the proteins, which were denatured at 98°C and stored in a refrigerator at −20°C. Colloidal preparation, electrophoresis, membrane transfer, sealing, and other operations were carried out successively according to the kit’s instructions. The following primary antibodies were used during incubation: goat anti-IL-1β polyclonal antibody (1:1000, R & D Systems, USA), rabbit anti-CaSR polyclonal antibody (6D4, 1:500, Santa Cruz Biotechnology, USA), rabbit anti-NLRP3 polyclonal antibody (1:500, Novus, USA), mouse anti-caspase-1 monoclonal antibody (1:500, Novus, USA), and mouse anti-β-actin polyclonal antibody (1:500, Boster, China). Protein bands were detected using ECL (Pierce, USA).
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