At 48 h after transfection, transfected SW620 cellular protein was extracted by a lysis buffer and quantified by a bicinchonininc acid kit (Beyotime, Shanghai, China). All the cellular protein lysates within this study were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Then, the membrane was incubated with specific primary antihuman antibodies followed by their respective appropriate secondary antibodies. The bands were visualized by electrochemiluminescence automatic chemiluminescence imaging analysis system (Tanon, Shanghai, China). A GAPDH antibody was used as a control, and the anti-Bcl-2, cleaved caspase-3 and cleaved caspase-9 (all 1:1,000) antibodies were purchased from Cell Signaling Technology, Inc. USA.
Bicinchonininc acid kit
Manufactured by Beyotime
Sourced in China
The Bicinchonininc acid kit is a colorimetric assay used for the quantitative determination of protein concentration. It utilizes bicinchoninic acid (BCA) to detect and quantify the total protein content in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Wbkls0100, by Merck Group (1 mentions)
Ab1377, by Abcam (1 mentions)
Ab195352, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Pa5 34430, by Thermo Fisher Scientific (1 mentions)
Ab8227, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
P8340, by Merck Group (1 mentions)
Gb23303, by Wuhan Servicebio Technology (1 mentions)
Gb23301, by Wuhan Servicebio Technology (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Hrp 60004, by Proteintech (1 mentions)
3 protocols using bicinchonininc acid kit
1
Quantifying Apoptosis-Related Proteins
2
Quantitative Protein Analysis in Cells
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Total protein in tissues or cells was extracted with a Radio Immunoprecipitation Assay lysis buffer solution containing phenylmethyl sulfonylfluoride. Cells were then centrifuged at a temperature of 4° C and at the rate of 8000 ×g for the duration of 10 minutes to harvest the supernatant. The bicinchonininc acid kit (P0012, Beyotime Institute of Biotechnology, China) was employed to measure the total protein concentration. Then, 50 μg of protein was dissolved in 2 × sodium dodecyl sulfate (SDS) loading buffer. After boiling both at 100° C for 10 min, each sample was subjected to SDS-polyacrylamide gel electrophoresis. The protein was transferred to a polyvinylidene fluoride (PVDF) membrane using wet transfer. After blocking the PVDF membrane for 1 h, it was incubated at 4° C overnight with diluted primary antibodies: rabbit anti-EED (1 : 1000, PA5-34430, Invitrogen), rabbit anti-METTL3 (1 : 1000, ab195352, Abcam, Cambridge, UK), rabbit anti-CDCP1 (1 : 1000, ab1377, Abcam), and murine anti-β-Actin (1 : 5000, ab8227, Abcam). This was followed by incubation with secondary antibody to immunoglobulin G (IgG) (Abcam, ab205718, goat anti-rabbit, 1 : 20000; Abcam, ab205719, goat anti-mouse, 1 : 20000) at room temperature for 1 h. The blots were developed with the enhanced chemiluminescence substrate (WBKLS0100, Millipore, Billerica, MA, USA) and quantified using the Image J software.
3
Western Blot Analysis of Protein Targets
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Cells or tissues were treated with Radio Immunoprecipitation Assay buffer (Beyotime, Shanghai, China) containing protease inhibitors (P8340, Sigma‐Aldrich, St. Louis., MO, USA). The concentration of proteins was measured by using a bicinchonininc acid kit (Beyotime). Extracted proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% non‐fat milk and incubated with primary antibodies for anti‐PKM2 (1:1000, ab154816, Abcam, USA), anti‐ALYREF (1:800, GTX113917, GeneTex, Irvine, CA, USA), anti‐HIF‐1α (1:1000, #36169, Cell Signaling Technology, USA), anti‐NSUN2 (1:1000, 702036, ThermoFisher, Waltham, MA, USA), anti‐GAPDH (1:1000, HRP‐60004, Proteintech, Rosemont, IL, USA), or anti‐β‐actin (1:1000, 66009‐1‐lg, Proteintech). The membrane was then incubated with anti‐mouse secondary antibodies (1:5000, GB23301, Servicebio, Wuhan, Hubei, China,) or anti‐rabbit secondary antibodies (1:5000, GB23303, Servicebio). Protein levels were detected using an enhanced chemiluminescence system (Pierce, Rockford, IL, USA).
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