The phosphatase activities of PP1 or PP2A in vehicle- or MC-LR–treated murine follicles and human primary granulosa cells were measured by using the RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Briefly, immature mouse follicles were cultured using eIVFG and treated with vehicle or MC-LR from day 2 to day 6 as we described above. On day 6 of eIVFG, 25–30 follicles were pooled together in each treatment group and lysed with the M-PER Mammalian Protein Extraction Reagent (ThermoFisher). The protein concentrations of follicle lysates were then determined using the Bradford protein assay kit (Abcam). Next, equal amounts of proteins ( ) in each group were used to determine the phosphatase activity of PP1 and PP2A. The plate containing loaded proteins was incubated at RT and the fluorescence was then measured at multiple time points on a Spectramax M3 microplate reader (Molecular Devices) with excitation and emission wavelengths of 355 and , respectively.
Bradford protein assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Bradford protein assay kit is a colorimetric assay used to determine the concentration of protein in a solution. It relies on the binding of Coomassie Brilliant Blue G-250 dye to proteins, which results in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ripa buffer, by Yeasen (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Rediplate 96 enzchek serine threonine phosphatase assay kit, by Thermo Fisher Scientific (1 mentions)
Spectramax m3 microplate reader, by Molecular Devices (1 mentions)
Pepsin, by Merck Group (1 mentions)
Sircol colorimetric assay, by Biocolor (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sds page, by Beyotime (1 mentions)
Enhanced chemiluminescence detection system, by Merck Group (1 mentions)
Ab91526, by Abcam (1 mentions)
Ab51520, by Abcam (1 mentions)
Ab202030, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab51772, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab92498, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
C myc, by Proteintech (1 mentions)
6 protocols using bradford protein assay kit
1
Measuring Phosphatase Activities in Follicles
2
Quantifying Fibrillar Collagen in Tissues
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Non-crosslinked fibrillar collagen in lung and skin samples was measured by the Sircol colorimetric assay (Biocolor, Belfast, UK). Minced tissues were homogenized in 0.5 M acetic acid with about 1:10 ratio of pepsin (Sigma, USA). Tissues were weighted and incubated overnight at 4 °C with vigorously stirring. Digested samples were centrifuged and the supernatant was used for Sircol dyeing to detect the collagen content. The total protein concentration was determined by using Bradford protein assay kit (Abcam) and the collagen content of each sample was normalized to total protein.
3
Protein Expression Analysis in HPAEpiCs and Lung Tissues
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The total proteins were extracted from HPAEpiCs or lung tissues by using lysis buffer (P0013G, Beyotime, China), and the protein concentration in each extract was determined using a Bradford protein assay kit (ab102535, Abcam, Cambridge, UK. Next, a 20 ug sample of total protein from each extract was separated by SDS-PAGE (10%; Beyotime), and the protein bands were transferred onto PVDF membranes (Millipore, Billerica, MA, USA), which were subsequently incubated overnight at 4°C with the following antibodies: anti-LC3B (1 : 1000, ab51520, Abcam, Cambridge, MA, USA), P62 (1 : 1000, ab91526, Abcam), Beclin-1 (1 : 1000, ab207612, Abcam), SLUG (1 : 1000, ab51772, Abcam), HIF-1α (1 : 500, ab92498, Abcam), E-cadherin (1 : 500, ab40772, Abcam), N-cadherin (1 : 1000, ab202030, Abcam), and GAPDH (1 : 10000, ab8245, Abcam). Next, the membranes were incubated with an HRP goat anti-rabbit/mouse IgG secondary antibody (1 : 20000, ab8245, Abcam); after which, the immunostained protein bands were detected using an enhanced chemiluminescence detection system (Millipore). The integral optical density values of the protein bands were analyzed using Image Pro Plus 6.0 software.
4
SDS-PAGE Analysis of Hemolymph Proteins
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Total protein concentrations were extracted from the samples using a Tris-buffer system prior to the colorimetrical control using the Bradford Protein Assay Kit (ab102535, Abcam, Cambridge, UK). The hemolymph total soluble proteins were qualitatively analyzed of employing sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli [68 (link)]. Briefly, protein samples of equal amounts (25 µg) were mixed with SDS sample buffer and boiled for 5 min, then ice-cooled for 7 min followed by SDS-polyacrylamide gel (15% resolving gel) and the vertical electrophoresis unit (Cleaver, UK) for the final ingredients separation. To estimate the molecular weights of the separated bands, a prestained molecular weight marker of the low range 180–10 kDa (Sigma, St. Louis, MO, USA) was loaded.
5
Western Blot Analysis of Cellular Proteins
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Cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer (Yeasen) containing a protease inhibitor cocktail (Sigma-Aldrich). Proteins were quantified by a Bradford Protein Assay Kit (Abcam), and the same amounts of proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Pall, Port Washington, NY, United States), followed by a block with 8% milk dissolved in Tris-buffered saline Tween for 1 h at room temperature. And then the membranes were incubated with specific primary antibodies at 4°C overnight, followed by secondary antibodies at room temperature for 1 h. Antibodies used were as follows: CTCF (1:5,000, Abcam), GAPDH (1:5,000, ProteinTech), WNT4 (1:1,000, Santa Cruz), β-catenin (1:1,000, Cell Signalling Technology), and c-Myc (1:5,000, ProteinTech).
6
Protein Isolation and Western Blot
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Cells were harvested and lysed in RIPA buffer (Yeasen, Shanghai, China) containing a protease inhibitor cocktail (Sigma-Aldrich, Massachusetts, USA). Proteins were quantified using a Bradford Protein Assay Kit (Abcam, MA, USA), and the same amounts of proteins were separated on 10% sodium dodecyl sulfate (SDS)-PAGE gels and transferred to nitrocellulose membranes, which were blocked with 8% milk dissolved in Tris-buffered saline and Tween for 1 h at room temperature. The membranes were then incubated with specific primary antibodies at 4 °C overnight, followed by an incubation with secondary antibodies at room temperature for 1 h. The antibodies used are listed in Supplementary Table 1.
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