After treatment with ponatinib for 36 h, cells were suspended in radio-immunoprecipitation assay (RIPA) cell lysis buffer and lysed by homogenization. The cell lysates were collected by centrifugation, and the protein concentration was quantified using the BCA protein quantitation kit (Boster Biotechnology, Wuhan, China). A total of 100 μg protein per sample was separated by SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked using 5% skim milk at room temperature for 1 h and incubated at 4 °C overnight with primary antibodies in 5% skim milk. Primary antibodies were detected with a corresponding HRP-conjugated IgG antibody, and bands were visualized using an enhanced chemiluminescent (ECL) blot detection system (Transgene, Beijing, China). Image J software was used to quantify the grey value of the bands.
Bca protein quantitation kit
Manufactured by Boster Bio
Sourced in China
The BCA protein quantitation kit is a colorimetric assay used for the determination of protein concentration. It is based on the bicinchoninic acid (BCA) method, which involves the reduction of copper(II) to copper(I) by protein in an alkaline medium, and the subsequent colorimetric detection of the copper(I) by bicinchoninic acid.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Ecl blot detection system, by Transgene (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Vegfa, by Abcam (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Phospho stat3 p stat3, by Cell Signaling Technology (1 mentions)
β actin, by R&D Systems (1 mentions)
2 protocols using bca protein quantitation kit
1
Ponatinib-Induced Protein Analysis
2
Protein Expression Analysis in Retinal and Monocyte Samples
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The protein of retinal tissue or bone marrow–derived monocytes was extracted using a radioimmunoprecipitation assay (RIPA) buffer and 1-mM phenylmethylsulfonyl fluoride, phosphatase inhibitors, and a protease inhibitor. The concentration of each specimen was determined using the BCA Protein Quantitation Kit (Boster Biological Technology, Wuhan, Hubei, China), and the samples were separated with SDS-PAGE and transferred to polyvinylidene difluoride filter membranes. The membranes were blocked with 5% bovine serum albumin in PBST (PBS containing 0.5% Tween) at room temperature for 1 hour and then incubated with primary antibodies overnight at 4°C. Expression of the STAT3 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), phospho-STAT3 (pSTAT3; 1:1000 dilution; Cell Signaling Technology), VEGFA (1:1000 dilution; Abcam, Cambridge, UK), and β-actin (1:10,000 dilution; R&D Systems) proteins in retinas with OIR at P17 and BMDMs was measured. The following day, the membranes were incubated with secondary antibodies for 1 hour at room temperature. An enhanced chemiluminescence kit (MilliporeSigma, Billerica, MA, USA) was used to detect the blots, and the bands were quantified using ImageJ software.
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