To prepare protein extracts, testes were homogenized in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Roche Diagnostics). After transient ultrasound treatment, the testis lysates were incubated on ice for 30 min and then centrifuged at 4 ℃, 12000 rpm for 20 min. The supernatant was transferred to a new tube and quantified using a BCA reagent kit (Beyotime, P0012-1). Then equal volume loading buffer was added. After being boiled at 95 ℃ for 10 min, the protein lysates were used for immunoblotting analysis. Immunoblotting was performed as described previously 45 (link). Briefly, the separated proteins in SDS-PAGE were electrically transferred to a polyvinylidene fluoride membrane. After incubation with primary and secondary antibodies, the membranes were scanned with Bio-Rad ChemiDoc XRS+.
Bca reagent kit
Manufactured by Beyotime
Sourced in China, United States
The BCA reagent kit is a colorimetric assay used for the quantitative determination of total protein concentration. It contains the necessary reagents to perform the BCA (Bicinchoninic Acid) protein assay.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
Ripa buffer, by Beyotime (3 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions)
Chemiluminescent hrp substrate, by Merck Group (3 mentions)
Quantity 5.2 software system, by Bio-Rad (3 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
β actin, by Abcam (2 mentions)
Chemiluminometer, by Bio-Rad (2 mentions)
Gapdh, by Proteintech (2 mentions)
Enhanced chemiluminescence kit, by Beyotime (2 mentions)
Cellytic mt cell lysis reagent, by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Cell lysis buffer, by Beyotime (2 mentions)
Pvdf membranes, by ABclonal (2 mentions)
Secondary antibodies conjugated with horseradish peroxidase, by ABclonal (2 mentions)
41 protocols using bca reagent kit
1
Protein Extraction and Immunoblotting from Testes
2
Western Blot Analysis of Cervical Cancer
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Total proteins were extracted from cervical cancer cells or tissues using RIPA buffer (Beyotime, Shanghai, China) containing proteinase inhibitors (Beyotime, Shanghai, China). The protein concentration was detected by the BCA reagent kit (Beyotime, Shanghai, China). 20 μg of protein was loaded and separated by 15% SDS-PAGE, and transferred to the PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk at RT for 1 h followed by incubating with the primary antibodies at 4ºC overnight. Subsequently, the HRP-conjugated secondary antibodies were added in and incubated for 1 h at RT. The signals were detected with the enhanced chemiluminescence kit (ECL, Millipore, Billerica, MA, USA) according to the manufacturer’s instruction. GAPDH was detected as the loading control.
3
Western Blot Analysis of Cellular Proteins
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Total proteins from the cells or tissues were extracted with RIPA lysis buffer containing proteinase inhibitor. The protein concentrations were measured by the BCA reagent kit (Beyotime). The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, which were blocked in 5% non-fat milk, and then incubated with the primary antibodies against ARID1A (Bethyl Laboratories), NF-κB p65 (RELA) (Santa Cruz), E-cadherin (Santa Cruz), CagA (Santa Cruz), p21 (Proteintech) and β-actin (Abcam) at 4 °C overnight. The membranes were then washed in tris buffered saline tween and incubated with anti-mouse or rabbit horseradish peroxidase-conjugated secondary antibodies and developed with the enhanced chemiluminescence method (ECL, Millpore). β-actin served as a loading control.
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells using RIPA lysis buffer (Sigma-Aldrich; Merck KGaA) containing protease inhibitors (Roche Diagnostics); the protein concentration was measured using a BCA reagent kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg/lane) were separated via 5-10% SDS-PAGE and transferred to PVDF membranes (Merck KGaA). Membranes were blocked with TBST with 0.1% Tween-20 containing 5% nonfat milk for 2 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: Anti-GATA6 (1:2,000; cat. no. 5851; Cell Signaling Technology, Inc.); anti-GAPDH (1:10,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.); anti-LOXL2 (1:2,000; cat. no. ab96233; Abcam); anti-VEGFA (1:2,000; ab46154; Abcam); and anti-Flag (1:1,000; cat. no. 14793, Cell Signaling Technology, Inc.). Membranes were washed with TBST for 30 min and incubated with an HRP-conjugated anti-rabbit secondary antibody (1:4,000; cat. no. ab6721; Abcam) for 2 h at room temperature. The immunocomplexes were visualized using the Immobilon™ Western Chemiluminescent HRP substrate (cat. no. WBKLS0100; Merck KGaA). ImageJ v1.51 software (National Institutes of Health) was used to quantify protein expression level.
5
Protein Extraction and Western Blot Analysis
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The total protein of cells was extracted by radio-immune precipitation assay (RIPA) lysis buffer adding protease inhibitors PMSF (Beyotime, China) at a ratio of 100:1 (v/v), the protein concentration was detected by BCA reagent kit (Beyotime). To denature protein, mixing the protein sample with 5× loading buffer at ratio of 4:1 (v/v), boiling the mixed solution at 100°C for 10 min. Then, 30 μg of total protein was loaded into the wells of SDS-PAGE gel to separate, after running time, the gel was transferred to polyvinylidene difluoride (PVDF) membrane and the proteins were detected by different antibodies including antibodies against EIF3C (Abcam, United States) and GAPDH (Proteintech, China). An anti-mouse horseradish peroxidase antibody was used as a secondary antibody. Finally, the PVDF membranes were imaged with the enhanced chemiluminescence reagent (ECL, Millipore) by a chemiluminometer (BioRad, United States).
6
Western Blot Analysis of Spinal Cord Proteins
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The spinal cords tissues were lysed in ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was determined using a BCA reagent kit (Beyotime Biotechnology, Shanghai, China). Total protein (30 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in tris-buffered saline with 5% non-fat milk and 0.5% bovine serum albumin for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies (1:1,000). After washing, the membranes were incubated with secondary antibodies (1:5,000) for 1 h at 37°C. Blots were visualized with the Chemiluminescent HRP substrate (Millipore) and quantified with the Quantity 5.2 software System (Bio-Rad).
7
Colon Pro-inflammatory Cytokine Assay
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Colon samples were homogenised in RIPA lysate (Thermo Fisher Scientific, USA), and the supernatant was collected for assessments of pro-inflammatory cytokines (n = 4 for each group). TNF and IL-6 were detected using the commercial ELISA kits (Biolegend, USA), according to the instruction from the manufacturer. The final results were represented as the ratio of cytokine concentration to total protein content, which was determined using a BCA reagent kit (Beyotime Institute of Biotechnology, China).
8
Protein Expression Analysis by Immunoblotting
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Total protein was extracted from cells using CelLyticTM MT Cell Lysis Reagent (Sigma, cat. C3228) and qualified using the BCA reagent kit (Beyotime, cat. P0011). Protein was loaded onto 10% gels for immunoblotting with antibodies against OR2T6, E-cadherin, N-cadherin, β-catenin, vimentin, H-RAS, c-Myc, ERK, c-fos, or β-actin separately. β-actin was used as the loading control. Detailed information about the antibodies was listed in Table 1 . The protein bands were visualized using an enhanced chemiluminescence kit (Beyotime, cat. P0018S).
9
Western Blot Protein Expression Analysis
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Cells were lysed in RIPA lysis buffer (BD Pharmingen, San Diego, CA, USA), protein concentration was measured by BCA reagent kit (Beyotime Institute of Biotechnology, Haimen, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate cellular lysates, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA, USA). The transfected membranes were blocked in 5% skim milk and incubated in 4°C overnight using the following primary antibodies: anti-ATF1 (1:1,000), anti-ATM (1:1,000), anti-ATM (phospho-1981; 1:5,000) (all from Abcam, Cambridge, MA, USA), anti-p53 (1:800), anti-p21 (1:500), anti-Bax (1:1,000) and anti-Bcl-2 (1:500) (all from Wanleibio, Shenyang, China), anti-GAPDH (1:3,000; Proteintech Group, Inc., Chicago, IL, USA). Then incubated with horseradish peroxidase-conjugated secondary antibody, goat anti-rabbit (1:5,000) or goat anti-mouse (1:5,000) (both from Cell Signaling Technology, Inc., Danvers, MA, USA). All bands were visualized with enhanced chemiluminescence (ECL) kit (Millipore).
10
Western Blot Protein Quantification
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Protein concentration was measured using a BCA reagent kit (Beyotime, Shanghai, China). Protein samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10% gel), and transferred onto polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was incubated with a primary antibody (anti-p65, anti-p-p65, anti-NLRP3, anti-pro-caspase-1, anti-ASC or anti-IL-1β) overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Protein bands were imaged by using the Omega Lum G imaging system (Aplegen, Pleasanton, CA).
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