Total proteins were extracted using RIPA lysis buffer (Beyotime) and protein concentration was determined using BCA Protein Assay Kit (Beyotime). Equal amounts of protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Nonspecific binding proteins were blocked with 5% non-fat milk for 1.5 h at room temperature. Subsequently, membranes were incubated overnight at 4̊C with antibodies against NCOA5 (Bioworld, BS67243, 1:1,000), Ki67 (Abcam, ab16667, 1:1,000), PCNA (Abcam, ab92552, 1:1,000), MMP2 (Abcam, ab92536, 1:1,000), MMP9 (Abcam, ab76003, 1:1,000), VEGFA (Thermo Fisher Scientific, OPA1-10110, 1:200), VEGFR2 (Abcam, ab134191, 1:1,000), TPX2 (Abcam, ab252945, 1:1,000) and GAPDH (Abcam, ab9485, 1:2,500). On the next day, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, ab6721, 1:3,000) for 2 h at room temperature. GAPDH served as the endogenous control. Enhanced chemiluminescence (ECL) kit was applied to develop the protein bands and the blots were visualized and analyzed by a Bio-Rad imaging system (Bio-Rad).
Ab134191
Manufactured by Abcam
Sourced in United Kingdom
Ab134191 is a lab equipment product manufactured by Abcam. It is a device designed for use in scientific research and laboratory settings. The core function of this product is to facilitate the performance of specific laboratory procedures or analyses, but a detailed description is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Ab92552, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab189494, by Abcam (1 mentions)
Ab211737, by Abcam (1 mentions)
Ab216880, by Abcam (1 mentions)
Ab205336, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab32536, by Abcam (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Ripa lysate, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
3 protocols using ab134191
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of HBMEC Signaling
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The cell lysates of HBMECs were made adopting RIPA buffer (Beyotime, Shanghai, China) with 1% protease and phosphatase inhibitor cocktail at 4°C and centrifuged. The supernatant was then collected for the protein concentration detection using Bradford Protein Assay Kit (Yubo Biological Technology, Shanghai, China). Protein in equal amounts was separated by 12% SDS-PAGE, followed by a transferring onto PVDF membranes. After blocking with 5% nonfat milk at room temperature for 1 h, the incubation of membranes was conducted with primary antibodies targeting VEGF (Beyotime, AV202, 1:1000), p-VEGFR2 (Bioworld, Y1175, 1:1000), VEGFR2 (Abcam, ab134191, 1:1000), ZO-1 (Abcam, ab216880, 1:1000), VE-cadherin (Abcam, ab205336, 1:1000), Claudin-1 (Abcam, ab211737, 1:2000), SIRT1 (Abcam, ab189494, 1:1000), FOXO3a (Abcam, ab109629, 1:1000), p-p65 (R&D, YB-22488, 1 mg/ml), p65 (Abcam, ab32536, 1:1000) overnight at 4°C. After washing with TBST for three times, the horseradish peroxidase-conjugated secondary antibody was added for 1 h incubation at room temperature. An enhanced chemiluminescence (ECL; Tanon, Shanghai, China) detection system was adopted to detect the protein bands. The quantification of protein bands were carried out with the aid of Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA).
3
Quantifying Protein Expression Levels
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Total protein of the cells was extracted by RIPA lysate (Sigma, Beijing Brand, China) and the protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Then 20 μg of total protein was taken for SDS-PAGE on 12% separating gel and 5% stacking gel, and transferred onto PVDF membranes. After blocking with PBST containing 5% skim milk for 1 h, the membranes were incubated overnight at 4 °C with mouse anti-FLT-1 (Proteintech, Wuhan, China, 13687-1-AP, 1:1000), rabbit anti-VCAM-1 (Abclonal, Wuhan, China, A0279, 1:500), rabbit anti-VEGFR-2 (Abcam, Cambridge, UK, ab134191, 1:1000) and mouse anti-GAPDH (Abcam, Cambridge, UK, ab8245, 1:5000), respectively, followed by washing three times with PBST. Then, the membranes were incubated with HRP-labelled goat anti-rabbit IgG or anti-mouse IgG (Proteintech, Wuhan, China, HRP-66467, HRP-67761, 1:10,000) for 2 h. After three washes with PBST, the membranes were immersed in ECL multifunctional substrate for 1 min, and the images were captured by the multifunctional imaging analysis system (FluorChem R, Protein simple, Silicon Valley, CA).
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