Protein was obtained from bovine preadipocytes using a protein extraction kit, supplemented with PMSF (Beyotime, Shanghai, China), and protein concentration was quantified with the Quantitative Protein Kit (the BCA method) (APPLYGEN, Beijing, China). For Western blot, 20 μg total cellular protein was separated on a 10% polyacrylamide gel, and then transferred onto PVDF. After blocking in Quick Block™ Western blocking solution (Beyotime, Shanghai, China) for 30 min at RT, the PVDF was sequentially incubated overnight at 4 °C with primary antibodies, respectively. Table 1 lists the dilution factor and company name of different antibodies. After washing in TBST-1× buffer 3 times (10 min each time) (Sangon Biotech Co., Ltd., Shanghai, China), the Goat anti-rabbit HRP antibody (absin, 1:10,000) diluted in Secondary Antibody Dilution Buffer (Beyotime, Shanghai, China) was incubated for 2 h, and washed in TBST-1× buffer.
Quantitative protein kit
Manufactured by Applygen
Sourced in China
The Quantitative Protein Kit is a laboratory equipment designed for the quantitative analysis of protein concentrations. It provides a standardized method for accurately measuring protein levels in samples. The kit includes all the necessary reagents and consumables required to perform the analysis.
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2 protocols using quantitative protein kit
1
Bovine Preadipocyte Protein Extraction and Western Blot
2
Protein Extraction and Western Blotting
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Briefly, proteins were extracted from specimens with the stents removed. Protein concentration was measured with a quantitative protein kit (Applygen, Beijing, China). An equivalent of 30 mg total protein was used for Western blotting analysis based on standard procedures. Primary antibodies, from the same source as for the immunohistochemical analysis, MMP-2 and MMP-9 were used. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mouse IgG1 GAPDH Monoclonal antibody, A01020 [Abbkine, Watlham, MA, USA]) protein was used as a loading control.
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