Total protein was extracted from kidney tissue using RIPA buffer (Servicebio, China) and detected with a BCA protein assay kit (Nanjing Jian Cheng Co., China) to determine the concentration of total protein. Protein was separated by SDS-PAGE, electrophoretically transferred to PVDF membranes, and then blocked in 5% nonfat milk. The protein bands were incubated at 4°C overnight with primary antibodies, including Bax, Bcl-2, caspase 3, and Cyto-c, followed by incubation with the secondary antibody for 2 hours at room temperature. Protein expression was detected using the Quantity One analysis system (BioRad, USA) and analyzed with ImageJ. β-Actin was used as an internal reference.
Quantity one analysis system
Manufactured by Bio-Rad
Sourced in United States
The Quantity One analysis system is a software application used for analyzing and quantifying data from various gel electrophoresis and imaging techniques. It provides tools for image acquisition, processing, and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Bca protein assay kit, by Keygen Biotech (2 mentions)
Hrp conjugated goat anti rabbit or rabbit anti mouse igg, by Beyotime (2 mentions)
Fluorescein linked secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Wuhan Servicebio Technology (1 mentions)
Bca protein assay kit, by Nanjing Jiancheng (1 mentions)
Ki 67 h 300, by Santa Cruz Biotechnology (1 mentions)
Pcna fl 261, by Santa Cruz Biotechnology (1 mentions)
β actin c4, by Santa Cruz Biotechnology (1 mentions)
β catenin antibody, by Cell Signaling Technology (1 mentions)
Nonfat milk, by BD (1 mentions)
Ab192396, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab2302, by Abcam (1 mentions)
Image station 4000, by Kodak (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Cell lysis buffer, by Keygen Biotech (1 mentions)
14 protocols using quantity one analysis system
1
Kidney Protein Expression Analysis
2
Western Blot Analysis of Cell Signaling Proteins
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The cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotech, Jiangsu, China), and centrifuged at 12000× g for 45 min at 4°C. After harvesting the supernatant, protein concentration was determined using the BCA assay kit KeyGEN (Beyotime Biotech). Samples containing 60 μg of protein were resolved on 12% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (Whatman International Ltd., Germany). After blocking for 1 h, the membranes were incubated with the following primary antibodies overnight at 4°C: Ki-67 (H-300), PCNA (FL-261), cyclin E (M20), cyclin D1 (H-295), p27 (C-19), p21 (C-19), CDK2 (D-12), CDK4 (C-22), CDK6 (C-21), p-ERK 1/2 (E-4), ERK 1/2 (H-72), p-JNK (G-7), JNK (D-2), p-AKT/Thr308 (p-AKT), AKT, and β-actin (C4): dilution 1: 200 (Santa Cruz Biotechnology) antibody. After being washed 3 times with TBST buffer, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 1.5 h at room temperature and analyzed using the Quantity One analysis system (Bio-Rad, Hercules, CA, USA).
3
Profiling Microbial Community Diversity
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PCR amplification and DGGE analysis were performed as previously described (16 (link)). Briefly, the variable V3 region of the 16S rRNA gene was amplified using the universal bacterial primers F357-GC and R518 with the sequences of 5′-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3′ and 5′-CCCGGGATCCAAGCTTACGGCTACCTTGTTACGACTT-3′, respectively. The PCR was performed as previously described. The amplified sequences of 16S rRNA were analyzed via DGGE fingerprinting analysis using 35 to 70% denaturing gel. Each lane uploaded 30 µl of PCR product, and electrophoresis was performed at 70 V for 990 min. Next, the DGGE gels were stained for 30 min with ethidium bromide (50 µg in 500 ml 1 X TAE buffer), and the band profiles were visualized and analyzed with a Quantity One Analysis System (Bio-Rad, California, USA). In short, all fingerprinting profiles were aligned using the reference lanes and then compared. The numeric value of the relative intensity per band class was then exported for all profiles and further analyzed. The Berger-Parker index, which was used to assess species richness, and α diversity measures, including the Shannon and Simpson diversity indices, were calculated based on the band profiles according to previously described method (17 (link)).
4
Quantitative Analysis of β-Catenin Signaling
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HUVECs from each group were added in RIPA lysis buffer (Beyotime, P0013B, China). Protein concentrations were measured using a BCA Protein Assay kit (KeyGEN Biotech, China). Proteins were separated on 10% SDS-polyacrylamide gels assayed by immunoblot using β-catenin antibody (Cell Signaling Technology, 8480S, USA), p-β-catenin (Ser675) antibody (Cell Signaling Technology, 9567S, USA), GSK3β (Ser9) antibody duet (Cell Signaling Technology, 8213S, USA), and β-actin antibodies (ZSGB-Bio, PR-0255, China). Secondary antibody used was (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (ZSGB-Bio, ZB-2305, ZB-2301, China) at 1:5000. Signals were visualized with super enhanced chemiluminescence (ECL) detection reagent (BOSTER, AR1172, China) and detected by Quantity One analysis system (Bio-Rad).
5
Western Blot Analysis of Apoptosis Markers
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HUVECs were collected and added with RIPA buffer (Beyotime, China) to extract the total protein. The concentrations of total protein were determined by using BCA method with a BCA Protein Assay Kit (KeyGEN Biotech, China). Proteins were then separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Next, the PVDF membrane was blocked in 5% nonfat milk (Becton Dickinson, USA) for 30 minutes at room temperature; followed by an overnight incubation at 4°C with primary antibodies, including cleaved caspase-3 (ab2302, Abcam, USA), PTEN (ab192396, Abcam, USA), and β-actin (ab8226, Abcam, USA). After the overnight incubation, PVDF membrane was washed for 3 times with TBST, and then the membrane was incubated with the secondary antibody for 2 hours at room temperature. The protein bands were quantified by using the ImageJ software, and the intensity of the protein bands was analyzed by the Quantity One analysis system (BioRad, USA). β-Actin was used as the internal control.
6
Western Blot Protein Detection
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Total protein was isolated from samples with lysis buffer. Proteins of interest were separated on SDS-PAGE gels, transferred to PVDF membranes (Millipore, Hong Kong, China), and incubated with Mst1 primary antibody (1:1000, Cell Signaling Technology, #3682) followed by horseradish peroxidase (HRP)-conjugated secondary antibody. The protein bands were detected by chemiluminescence (ECL) and were visualized using a Kodak Image Station 4000 (Rochester, NY). Band densities were quantified using the Quantity One analysis system (Bio-Rad Laboratories, UK) [63 (link)].
7
Western Blot Analysis of Signaling Pathway
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The expression of the target protein was visualized by western blotting. Cells were collected 2 h after drug intervention and total protein was extracted using cell lysis buffer (KeyGEN, Nanjing, China). A portion (50 μg) of each sample was loaded on 10% differential SDS-polyacrylamide gels and separated by the application of a continuous voltage. After electrophoresis, the protein was transferred to a PVDF membrane according to the molecular weight. Then, each membrane was blocked with 5% skim milk. The membranes were next incubated with different primary antibodies (Cell Signaling Technology, USA; dilution 1:1000): p70 S6 kinase and phospho-p70 S6 kinase (Thr389); p44/42 MAPK (Erk1/2) (137F5) and phospho-p44/42MAPK (Erk1/2) (Thr202/Tyr204) (197G2); Akt antibody, phosphor-Akt (Thr308) and phosphor-Akt (Ser473); c-Raf antibody and phospho-c-Raf (Ser338) (56A6); and β-actin at 4°C overnight. Corresponding horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA) was incubated with the membranes for 2 h at room temperature. The densities of bands were quantified using the Quantity One analysis system (Bio-Rad, Hercules, CA, USA).
8
Strontium Regulates HIF-1α, SDF-1, and VEGF in OVX-BMSCs
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OVX-BMSCs or HUVECs were seeded in 6-well plates (2×105 cells/well). OVX-BMSCs were cultured in medium supplemented with 100μM CoCl2 and different concentrations of SrR (0, 0.125, 0.25, 0.5, 1.0 and 2.0mM) for 48h. HUVECs were cultured in medium with SrR at 0.25mM for 0, 15, 30, 60 and 90 min. Then cells were washed by cold PBS, and lysed with RIPA for protein extraction. Total protein concentration was measured by a BCA protein assay kit.
Equal amount of protein samples were separated by 12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk for 1h, the membrane was incubated with mouse anti-rat HIF-1α (1:1000, CST, USA), SDF-1(1:200, Abcam, UK), VEGF (1:1000, Abcam, UK) and β-actin (1:5000, Sigma, USA) for OVX-BMSCs, rabbit anti human AKT, phosphorylated-AKT (p-AKT), mTOR, phosphorylated -mTOR (p-mTOR) (1:1000, CST, USA) for HUVECs at 4 °C overnight. Then followed by a 2h incubation with HRP-conjugated goat anti-rabbit or rabbit anti mouse IgG (1:3000, Beyotime, China) at room temperature and washed by PBST for 3 times. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) reagent (Pierce, USA), visualized by autoradiography and quantified by the Quantity One analysis system (Bio-Rad, USA). β-actin served as the internal control.
Equal amount of protein samples were separated by 12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk for 1h, the membrane was incubated with mouse anti-rat HIF-1α (1:1000, CST, USA), SDF-1(1:200, Abcam, UK), VEGF (1:1000, Abcam, UK) and β-actin (1:5000, Sigma, USA) for OVX-BMSCs, rabbit anti human AKT, phosphorylated-AKT (p-AKT), mTOR, phosphorylated -mTOR (p-mTOR) (1:1000, CST, USA) for HUVECs at 4 °C overnight. Then followed by a 2h incubation with HRP-conjugated goat anti-rabbit or rabbit anti mouse IgG (1:3000, Beyotime, China) at room temperature and washed by PBST for 3 times. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) reagent (Pierce, USA), visualized by autoradiography and quantified by the Quantity One analysis system (Bio-Rad, USA). β-actin served as the internal control.
9
Western Blot Analysis of IMD Protein
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Western blot analysis was performed as previously described.15 The primary antibody, rabbit monoclonal anti-rat IMD antibody (Phoenix Biotech, Beijing, P.R. China), was added at a dilution of 1:3000. Then, the fluorescein-linked secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added at a dilution of 1:3000. The specific bands were visualized by fluorography using an enhanced chemiluminescence kit (Pierce, Rockford, USA). The relative density was quantified using the Quantity One analysis system (Bio-Rad, California, USA).
10
Western Blot Analysis of Inflammatory Proteins
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The treated cells were removed from the culture media and extracted with the RIPA lysis buffer from Beyotime Biotech (Jiangsu, China) for 30 min. Supernatants were collected after the tubes were centrifuged at 10000 g for 40 min at 4°C. The protein concentrations were determined using a BCA Protein Assay Kit from Wuhan Boster Biological Technology (Wuhan, China). Samples containing 50 μg of protein were resolved by 12% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (Whatman International Ltd., Maidstone, Germany). Nonspecific binding was blocked by immersing the membranes into 5% nonfat dried milk and 0.1% (v/v) Tween 20 in PBS for 3 h at room temperature. After rinsing with a washing buffer (0.1% Tween 20 in PBS) several times, the membranes were incubated with a primary antibody against iNOS at 1 : 1000 dilution (catalog no. ab49999, Abcam) or an antibody against IL-1β at 1 : 1000 dilution (catalog no. ab150777, Abcam) overnight at 4°C. The membranes were washed several times, then incubated with a corresponding anti-mouse secondary antibody IgG conjugated to HRP (Cell Signaling Technology, Danvers, MA) at room temperature for 3 h, and analyzed by the Quantity One analysis system (Bio-Rad, Hercules, CA, USA). GAPDH at a dilution of 1 : 2000 (catalog no. ab 9483, Abcam) was used as an internal loading control.
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