RIPA buffer (Beyotime Institute of Biotechnology) containing PMSF (Beyotime Institute of Biotechnology) was used to extract cellular proteins. Protein concentration was assessed using a BCA protein analysis kit. Protein samples from each group (30 µg/lane) were separated using 10% SDS-PAGE and transferred onto PVDF membranes. PVDF membranes were blocked with QuickBlock™ Western rapid sealing solution (cat. no. P0252, Beyotime Institute of Biotechnology,) at room temperature for 30 min. Primary rabbit anti-VEGFA (1:1,000; cat. no. 50661; Cell Signaling Technology, Inc.) and anti-GAPDH (1:8,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.) antibodies were then added to the membranes, which were incubated at 4°C for ≥12 h. The membranes were incubated at 37°C for 1 h with HRP-labelled secondary antibody (1:3,000, cat. no. PR30011; ProteinTech Group, Inc; http://www.ptgcn.com/ ). Finally, a fusion imaging system QuantityOne (Bio-Rad Laboratories, Inc.) was used to assess the relative density of the band. ImageJ software (National Institutes of Health, version 1.8.0) was used for analysis.
Hrp labelled secondary antibody
Manufactured by Proteintech
Sourced in China
The HRP-labelled secondary antibody is a laboratory reagent used for immunodetection methods. It consists of a secondary antibody that is conjugated with the enzyme horseradish peroxidase (HRP). The HRP label enables signal amplification and can be detected through a colorimetric or chemiluminescent reaction, allowing for the visualization and quantification of target proteins in various immunoassay techniques.
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Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab184699, by Abcam (1 mentions)
Ab201015, by Abcam (1 mentions)
Enhanced chemiluminescence detection system, by Bio-Rad (1 mentions)
2 protocols using hrp labelled secondary antibody
1
Western Blot Analysis of VEGFA Expression
2
Protein Extraction and Western Blot Analysis
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Total proteins from cells were extracted using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations of all groups were determined by a BCA protein assay kit (Thermo Fisher Scienti c, Waltham, MA, USA). Proteins were separated by 10% SDS-PAGE, then transferred to PVDF membranes and incubated with speci c primary antibodies against t-ERK (ab184699, Abcam, Cambridge, UK), p-ERK (ab201015, Abcam, Cambridge, UK), and β-actin (10068-1-AP, Proteintech, Wuhan, China) overnight at 4°C. The membrane was incubated with HRP-labelled secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature. An enhanced chemiluminescence detection system (BIO-RAD, California, USA) was used to detect the protein bands.
Enzyme-Linked Immunosorbent Assay (ELISA)
Following appropriate CSE treatment and indicated transfection, the concentrations of interleukin-1β (IL-1β) and IL-6 from the culture supernatants of HBE cells were assayed using commercial the ELISA kits (Proteintech, Wuhan, China) referring to the manufacturer's protocols. The results represent as the average of three independent replicates.
Enzyme-Linked Immunosorbent Assay (ELISA)
Following appropriate CSE treatment and indicated transfection, the concentrations of interleukin-1β (IL-1β) and IL-6 from the culture supernatants of HBE cells were assayed using commercial the ELISA kits (Proteintech, Wuhan, China) referring to the manufacturer's protocols. The results represent as the average of three independent replicates.
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