Cells were lysed with RIPA buffer (Beyotime) containing protease inhibitors (04693132001, complete EDTA-free, Roche, Basel, Switzerland), and the supernatant was collected by centrifugation at 4 °C. The concentrations of proteins were quantified using the BCA assay kit (P0010, Beyotime, Shanghai, China), and 30 μg of protein from each sample was separated on a 12% SDS-PAGE gel. The proteins were transferred to a PVDF membrane, which was blocked in 5% bovine serum albumin (BSA) for 2 h at room temperature. The PVDF membrane was incubated with the primary antibodies (a list of specific antibodies is provided in Table S2 ) overnight at 4 °C. Secondary antibody conjugated to horseradish peroxidase was incubated with the PVDF membrane for 1 h at room temperature, and target proteins were detected by enhanced chemiluminescence (ECL) reagent (Applygen, Beijing, China). The bands were imaged by an imaging system (Sagecreation, Beijing, China), and quantified with Quantity One software (Bio-Rad, Hercules, CA, USA).
Imaging system
Manufactured by Sagecreation
Sourced in China
The Imaging System is a high-performance laboratory equipment designed for advanced imaging applications. It features a sophisticated camera and optics system that enables detailed and precise visual analysis of samples. The core function of the Imaging System is to capture and process images with exceptional clarity and resolution, providing users with valuable data for their research or diagnostic needs.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Beyotime (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Ecl reagent, by Applygen (1 mentions)
Mouse anti β actin, by Thermo Fisher Scientific (1 mentions)
Mouse anti histone h3, by Beyotime (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by Cyanagen (1 mentions)
Rabbit anti acetyl lysine, by Abcam (1 mentions)
Rabbit anti hdac1, by ABclonal (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
2 protocols using imaging system
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of PEDV Infection
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The IPEC-J2 cells infected with PEDV at appropriate MOIs or transfected with recombinant expression plasmids or siRNA were used for total protein extraction using the cell lysis buffer (Beyotime, China). The protein concentration of the whole-cell lysates in different treatments was measured by a bicinchoninic acid (BCA) protein assay kit (Thermo). Afterward, the heat-denatured protein samples were loaded onto 12% SDS-PAGE gels for protein fractionation followed by blotting onto a 0.22-μm polyvinylidene difluoride membrane (PVDF; Millipore). The proteins on the blots were then probed by specific primary and secondary antibodies, including rabbit anti-STAT1 (CST), rabbit anti-phospho-STAT1 at Y701 (ABclonal), rabbit anti-acetyl-lysine (Abcam), rabbit anti-acetyl-H3-K27 (ABclonal), rabbit anti-HDAC1 (ABclonal), rabbit anti-ISG15 (Beyotime), mouse anti-β-actin (Thermo), mouse anti-histone H3 (Beyotime), rabbit anti-HA tag (CST), mouse anti-PEDV N (monoclonal antibody reserved in our laboratory), goat anti-mouse IgG (Invitrogen) and goat anti-rabbit IgG (Invitrogen). The target protein bands on the membrane were developed using an ECL kit (Cyanagen, Italy) and were visualized by an imaging system (SageCreation, Beijing, China) for densitometric analysis of their relative levels of expression.
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