Bca reaction

Manufactured by Thermo Fisher Scientific

The BCA reaction is a colorimetric assay used for the quantitative determination of total protein concentration. It relies on the reduction of copper ions (Cu2+ to Cu1+) by proteins in an alkaline medium, and the subsequent chelation of the cuprous cations (Cu1+) with bicinchoninic acid (BCA) to produce a purple-colored complex that absorbs light at 562 nm. The intensity of the color is proportional to the protein concentration in the sample.

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Lab products found in correlation

Irdye 800cw goat anti rabbit secondary antibody, by LI COR (1 mentions) Nitrocellulose membrane, by GE Healthcare (1 mentions) Odyssey blocking buffer pbs, by LI COR (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Complete and phosstop, by Roche (1 mentions)

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BCA reaction buffer (2 citations)

2 protocols using bca reaction

Quantitative Western Blot Analysis

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After puromycin selection, cells were harvested and lysed using RIPA
buffer (50 mM Tris pH 8.0, 1mM EDTA, 150 mM NaCl, 1% Triton X-100,
0.1 % SDS, 0.1% sodium doxycholate) supplemented with
cOmplete proteinase inhibitor cocktail (Roche, Cat# 11697498001).
Lysate was homogenized using sonication and quantified using BCA reaction
(Thermo Fisher, Cat#23227). 20 μg of lysate was resolved on
NuPAGE 4-12% Bis-Tris gel (Thermo Fisher, Cat# NP0322) and
transferred to nitrocellulose membrane (GE Healthcare,
Cat#10600002). After blocking using Odyssey blocking buffer-PBS
(LI-COR BioSciences, Cat#927-40003), membrane was incubated for
overnight at 4°C with antibody against to MYC (Cell Signaling,
Cat#5605S, 1/1,000) or GAPDH (Santa Cruz, Cat#sc69778,
1/5,000). Afterward, the membrane was incubated for 1 hour at room
temperature with IRDye 800CW goat anti-rabbit secondary antibody (LI-COR
BioSciences, Cat#926-32211, 1/10,000) and IRDye 680RD goat
anti-mouse secondary antiboby (LI-COR BioSciences, Cat#926-68070,
1/10,000). Protein bands were visualized and quantified using Odyssey CLx
and Image Studio 1.0.11 (LI-COA BioSciences).

Cho S.W., Xu J., Sun R., Mumbach M.R., Carter A.C., Chen Y.G., Yost K.E., Kim J., He J., Nevins S., Chin S.F., Caldas C., Liu S.J., Horbeck M., Lim D.A., Weissman J.S., Curtis C, & Chang H.Y. (2018). Promoter of lncRNA gene PVT1 is a tumor suppressor DNA boundary element. Cell, 173(6), 1398-1412.e22.

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Western Blot Protein Analysis Protocol

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Cells for western blot experiments were replated the night before collection at sub confluent density, starved for 2 h for investigated cytokine, rinsed once with DMEM-F12 before treatment. Cells were lysed on ice with RIPA buffer containing protease and phosphatase inhibitors (Complete and PhosSTOP, Roche). Samples were centrifuged for 3 min at 15000 × g to remove cell debris and treated with DNAse to degrade genomic DNA. Protein concentration was determined by BCA reaction (Thermo) according to the manufacturer’s instructions. Equal amounts of protein (15–50 μg) were loaded in equal volumes to SDS-Polyacrylamide gradient gels. Proteins were transferred to nitrocellulose membranes and probed with indicated antibodies. The same membrane was stripped and probed with various antibodies unless otherwise stated. Details of antibodies are listed in Supplementary Table 3.

Varga B.V., Faiz M., Pivonkova H., Khelifi G., Yang H., Gao S., Linderoth E., Zhen M., Karadottir R.T., Hussein S.M, & Nagy A. (2022). Signal requirement for cortical potential of transplantable human neuroepithelial stem cells. Nature Communications, 13, 2844.

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