Protein samples were prepared by adding 200 μL of ice-cold NP-40 buffer with protease inhibitor to 200 μL of extracted EVs sample suspended in appropriate buffer. Mix and shake once every ten minutes then incubate on ice for 50 min. The protein concentration was measured by using BCA (Applygen, China). Then add 2 × loading buffer (200 μL), and mix well, and heat to boil. The protein sample (50 μg) was separated with an 8–10% SDS gel, and blotted on an immuno-blot PVDF membrane. Then the BSA was applied to block for 1 h and the membrane was incubated with primary antibody (APOB, AGO2, HSA, Alix, Tsg101, CD9 or CD63) overnight at 4 °C. The membrane was then washed with Tris buffered saline containing 0.1% Tween and incubated with the secondary antibody for 1 h at room temperature. Then, the film was washed again and exposed to ECL (electrochemiluminescence).
Bicinchoninic acid (bca)
Manufactured by Applygen
Sourced in China
The BCA (Bicinchoninic Acid) is a laboratory equipment used for quantitative determination of protein concentration. It is a colorimetric assay that relies on the reduction of copper ions by proteins in an alkaline environment, resulting in the formation of a purple-colored complex that can be measured using a spectrophotometer.
Automatically generated - may contain errors
Lab products found in correlation
Qam cyt 1 1, by RayBiotech (1 mentions)
Rifampicin rif, by Merck Group (1 mentions)
Human umbilical vein endothelial cells huvecs, by American Type Culture Collection (1 mentions)
Recombinant human tnf α, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
A21010, by Abbkine (1 mentions)
Ecl system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
4 protocols using bicinchoninic acid (bca)
1
EV Protein Profiling by Immunoblot
2
Colon Inflammation Analysis in Mice
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After the mice were subjected to 5% DSS for 5 days, 1 cm of the mouse proximal end of colon was added to 0.5 mL of tissue lysate (RIPA lysate and protease inhibitor; Applygen Technology Inc., Beijing, China). After homogenization and centrifugation at 12,000 × g, the supernatant was collected, and the total protein concentration was measured by BCA (Applygen Technology Inc., Beijing, China). Myeloperoxidase (MPO) levels were detected using an MPO ELISA kit (RayBiotech, Guangzhou, China), and cytokine levels were detected using a protein chip (QAM-CYT-1-1, RayBiotech, Guangzhou, China).
3
Tanshinone IIA Regulation of PXR Signaling
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Tanshinone IIA (>98% purity) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Human umbilical vein endothelial cells (HUVECs) and a human acute monocytic leukemia cell line (THP-1) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Roswell Park Memorial Institute 1640 medium (RPMI-1640) was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from GenStar (Beijing, China). Rifampicin (RIF) and human recombinant TNF-α were obtained from Sigma-Aldrich (St. Louis, MO, USA). GAPDH and rabbit histone polyclonal antibody was purchased from TDY Biotech (Beijing, China). Immobilon Western Chemi-Luminescent HRP Substrate, and rabbit monoclonal PXR antibody were purchased from Millipore Corporation (Millipore, CA, USA). BCA was purchased from Applygen Technologies Inc (Beijing, China). All other chemicals were of the highest quality commercially available.
4
Protein Extraction and Western Blot Analysis
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Cells were lysed with 100 μL RIPA lysis buffer (Applygen, Beijing, China) on ice for 60 min and then centrifuged at 12,000 rpm for 20 min at 4°C to pellet protein. The protein pellet was resuspended in 20μL PBS. Protein concentration was measured by the BCA (Applygen, China) protein determination method. The same amount of total protein (40 μg) was loaded onto 6% or 10% polyacrylamide gels for sodium dodecyl sulfate‒polyacrylamide (SDS-PAGE) gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes (PVDF). PVDF membranes were blocked with 5% BSA for 2 h and then incubated with diluted primary antibodies overnight at 4°C. Primary antibodies are listed in supplementary Table 2 . After washing three times with TBST, the PVDF membranes were incubated with the secondary antibody (A21010, Abbkine, Wuhan, China) at room temperature for 1 hour. Proteins were detected by an ECL system (Bio-Rad, USA) and analyzed using ImageJ (1.5.3) to determine abundance. Each experiment was repeated three times.
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