The kidney tissue (100 mg) was homogenized on ice in 1 mL RIPA (Beyotime Biotechnology, Shanghai, China) containing 1% PMSF and 2% phosphatase inhibitor, stewed and then centrifuged at 12000 rpm for 10 min at 4 °C. The supernatants were collected. The protein lysate concentration was measured for protein concentration. After using the BCA protein concentration determination kit (Beijing Dingguo Changsheng biotech CO. LTD, Beijing, China) to determine the protein concentration, proteins (20 μg) were resolved by 10% SDS-polyacrylamide electrophoresis gel electrophoresis (SDS-PAGE) which was followed by a transfer of proteins from gels to the nitrocellulose filter membranes. Next, the membranes were incubated with primary antibodies overnight at 4 °C, including TGF-β1 (A2124, 1:1000), Smad2 (A7699, 1:1000), p-Smad2 (18338, 1:1000), Smad3 (A19115, 1:1000), p-Smad3 (9520, 1:1000), Smad7(A16396,1:1200) and β-actin (AC026, 1:1000). The above-mentioned primary antibodies were obtained from ABclonal (ABclonal Technology Co., Ltd., Wuhan, China) except p-Smad2 and p-Smad3 which were from Cell Signaling Technology (CST, Danvers, MA, United States). The membrane was incubated with the appropriate secondary antibodies coupled to horseradish peroxidase and the blots were developed with the chemiluminescence reagent using an enhanced chemiluminescence kit (Biosharp, Hefei, China).
Bca protein concentration determination kit
Manufactured by Dingguo
Sourced in China
The BCA protein concentration determination kit is a laboratory equipment used to measure the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) method, which allows for the colorimetric detection and quantification of total protein. The kit provides a simple and reliable way to determine protein levels in a wide range of samples.
Automatically generated - may contain errors
Lab products found in correlation
P smad2, by Cell Signaling Technology (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Biosharp (1 mentions)
Hrp labeled goat anti rabbit secondary antibody, by Proteintech (1 mentions)
P stat3, by ABclonal (1 mentions)
Stat3, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
3 protocols using bca protein concentration determination kit
1
Western Blot Analysis of TGF-β1/Smad Pathway
2
Western Blot Analysis of Stem Cell Markers
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Western blotting was used to detect the target protein in the sample. The total protein content in the sample was detected in a 96-well plate using a BCA protein concentration determination kit (Dingguochangsheng). Protein tracer sample buffer (reduction, 5×; CWBIO) was mixed with protein samples in a ratio of 1:4. The mixtures were then placed in a boiling water bath for 3 minutes. The samples were cooled to room temperature and centrifuged at 13 000×g at 4 °C for 30 seconds. Denatured proteins were directly loaded to a sodium dodecyl sulfate-page gel, and conventional electrophoresis (concentration gel voltage 60 V, separation gel voltage 120 V) and membrane transfer (current 200 mA) were performed using a Bio-Rad protein imprinting device. After completion, the transferred films were incubated at 4 °C overnight. The rabbit primary antibodies used were STAT3 (Proteintech), pSTAT3 (Tyr705; ABclonal), KLF4 (Proteintech), OCT4 (Proteintech), and β-actin (Proteintech). Samples were incubated with HRP-labeled goat anti-rabbit secondary antibody (Proteintech) at room temperature for 1 hour, washed with 1× TBST 3 times for 10 minutes each, and finally ECL development was performed.
3
Termite Biochemical Responses to Predators
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Three days post-injection, termite individuals were weighed and then immediately crushed in liquid nitrogen and dissolved at a ratio of 10 mg of body fresh weight to 100 μL of the solution recommended by the manufacturer. Termite brains were dissected on ice immediately after foraging assays in different social contexts including predator ants (−) + nestmate soldiers (−), predator ants (+) + nestmate soldiers (−), and predator ants (+) + nestmate soldiers (+) for 15 min (“−” in parentheses means “not present” and “+” in parentheses means “present”), and then crushed in liquid nitrogen and dissolved in the solution recommended by the manufacturer. The determination of the ATP (N = 9 replicates), NADH (N = 6 replicates), IDH (N = 6 replicates), and glucose (N = 9 replicates) contents of the whole organism and the ATP (N = 6 replicates) and IDH (N = 6 replicates) contents of the brain was performed according to the protocols provided by the manufacturers (Beyotime Biotechnology [Shanghai, China], Nanjing Jiancheng Bioengineering Institute [Jiangsu, China], and Zeye Biological Technology [Shanghai, China]). The protein concentration of the brain was determined according to the protocol of the bicinchoninic acid (BCA) protein concentration determination kit from Beijing Dingguo Changsheng Biotechnology (Beijing, China).
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