Rat endometrium tissues were initially homogenized and lysed in radio-immunoprecipitation assay (RIPA) buffer (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China) supplemented with proteinase inhibitor (Lot#4693116001, Roche). Lysates were then centrifuged for 20 min at 12,000 g. The bicinchoninic acid (BCA) (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China) was used for quantification of protein concentration. Equivalent amounts of protein (20 μg) were then used for western blot with primary antibodies of VEGF (ab53465, Abcam, UK), PI3K (ab191606, Abcam, UK), P-PI3K (ab182651, Abcam, UK), AKT (ab8805, Abcam, UK), P-AKT (ab38449, Abcam, UK), Ang-1 (ab102015, Abcam, UK), Ang-2 (ab155106, Abcam, UK) and HIF-1a (ab1, Abcam, UK) at 4 °C with gentle shaking overnight. After washing with TBS-T for three times, the membranes were then incubated with the secondary antibody at RT for 1 h and detected using an ECL plus kit (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China). The ECL signals were detected with Quantity One software (Bio-Rad, Hercules, CA) and blot intensities were quantified by using imageJ (NIH, Bethesda, MD) software. Tubulin (ab8245, Abcam, UK) was used as an internal control to validate the amount of protein loaded onto the gels.
Ab102015
Manufactured by Abcam
Sourced in United Kingdom
Ab102015 is a monoclonal antibody designed for use in laboratory research. It targets a specific protein of interest, providing researchers with a tool to investigate its expression and function. The core function of this product is to facilitate the detection and analysis of the target protein through immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab9485, by Abcam (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Ab191606, by Abcam (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab53465, by Abcam (1 mentions)
Ab155106, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab182651, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ripa buffer, by Dingguo (1 mentions)
Ab9512, by Abcam (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Ab181603, by Abcam (1 mentions)
Ab6881, by Abcam (1 mentions)
Ab15086, by Abcam (1 mentions)
Ab128086, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
6 protocols using ab102015
1
Protein Expression Analysis of Rat Endometrium
2
Dual Immunolabeling of AQP4 and ANGPT1
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Immunolabeling was performed using the same protocol as was described above for NOTCH1 (single labeling). Primary antibody used were mouse monoclonal antibody synthetic AQP4 (1:100, #ab9512, Abcam) and rabbit-polyclonal antibody raised against human ANGPT1 (1:200, ab102015, Abcam). Specificity of antibodies has been demonstrated previously [29 (link)–30 (link)].
3
Protein Expression Analysis Protocol
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The primary antibodies used in this study were against the following proteins: PADI4 (ab128086, Abcam, Cambridge, UK); GAPDH (ab181603, Abcam, Cambridge, UK); carbonic anhydrase 9 (CA9) (ab15086, Abcam); angiopoietin 1 (ANGPT1) (ab102015, Abcam); tyrosine‐protein kinase receptor TIE2 (TEK) (NBP1‐69753SS, NovusBiologicals, Littleton, CO); and His‐tag (12698S, CST, Danvers, MA). The secondary antibodies used in this study were as follows: goat anti‐rabbit antibody: (#s0001, Affinity Biosciences, Cincinnati, OH); goat anti‐mouse antibody: (#s0002, Affinity Biosciences, Cincinnati, OH); donkey anti‐goat antibody: (ab6881, Abcam); goat anti‐rabbit IgG (H + L) highly cross‐adsorbed secondary antibody, Alexa Fluor 488 (A‐11304, Thermo Fisher Scientific, Waltham, MA); and goat anti‐mouse IgG (H + L) highly Cross‐Adsorbed, Alexa Fluor 568 (A‐11301,Thermo Fisher Scientific).
4
Western Blot Analysis of Ang1 Protein
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Primary hepatocyte cell lysate was prepared in ice-cold RIPA lysis buffer. Protein content was quantitated with the BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA, 23227) following the manufacturer’s guide. Protein was resolved by 10% SDS-PAGE gel and then transferred onto PVDF membrane (100V, 2h). After transfer the blots were blocked with 5% non-fat milk dissolved in the TBST buffer for 1h at room temperature with shaking. The Ang1 primary antibody (ab102015, 1:1500, Abcam) and Anti-GAPDH (ab9485, 1:2000, Abcam) were hybridized overnight at 4 °C. The blots were then washed with TBST and then the secondary antibody was added (Rabbit IgG HRP; 170-6515, 1:5000, Bio-Rad) and incubated for one hour at room temperature. The protein blot was then visualized using the Pierce ECL Western Blotting Substrate (32106, Thermo Fisher, Waltham, MA, USA). The analysis of the intensity of the signal was quantitated using ImageLab (Bio-Rad). The values of each band were normalized by dividing them by the value of their GAPDH and normalized to the control sample, which was assigned a value of 1 (Figure S7 ).
5
Western Blot Analysis of Angiogenic Factors
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The samples were homogenized and total proteins extracted using a radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Haimen, China). A BCA kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration. The proteins were loaded on sodium dodecyl sulfate (SDS) polyacrylamide gels (10%) (Aspen), transferred onto nitrocel-lulose membranes (Pall Corp., New York, NY, USA), and blocked in non-fat dry milk (5%) at room temperature to set for 2 h. The membranes were incubated overnight at 4°C with primary antibodies against Ang-1 (1:1,000; ab102015; Abcam), p-Tie-2 (1:1,500; sc-9026; Santa Cruz Biotechnology, Inc.), α-SMA (1:2,000; ab32575), collagen type IV (1:1,500; ab6586) and GAPDH (1:5,000; ab37168) (all from Abcam). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Aspen) for 1 h, and finally the membranes were detected using an enhanced chemiluminescence substrate (Beyotime Institute of Biotechnology).
6
Quantitative Western Blot Analysis of Angiogenic Factors
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Stomach tissues were macerated and lysed using lysis buffer solution (PBS 1X, 1% Triton X-100, 1% NP-40, proteases and phosphatases cocktail inhibitors, pH 7.4). Following centrifugation at 14 000 g for 30 min, the proteins were quantified using the Protein Assay Dye Reagent Concentrate (BioRad) and 50 µg of protein were separated by 10% SDS−PAGE, prior to being transferred onto a nitrocellulose membrane (BioRad). Following blocking with 5% fat−free milk in PBS buffer in 0.05% Tween 20 for 1 h at room temperature, the membranes were then separately incubated overnight at 4˚C with the following monoclonal antibodies: Rabbit ANGPT1 (1:500; Abcam ab102015), Rabbit ANGPT2 (1:500, Abcam ab8452) and Rabbit GAPDH (1:1000, Abcam, ab9485). The secondary antibodies were applied (dilution 1:10 000, horseradish peroxidase-conjugated anti-rabbit, Sigma-Aldrich A9169) at room temperature for 1 h. Labeled bands were detected by enhanced chemiluminescence (Clarity ECL, BioRad, USA) and analyzed by Chemidoc Imaging System (Bio-Rad). Protein levels were quantified using the Image Lab software, version 4.1 (Bio-Rad, USA).
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