The cells were washed three times in PBS and lysed directly using cell lysis buffer (50 mM Tris, 200 mM NaCl, 1% NP-40, pH 7.5) for 1 h at 4°C. 100× protease inhibitor cocktail (B14001; Biomake) was added to the lysis buffer before use. Lysates were centrifuged at 4°C at maximum speed (15,000 g) for 10 min. The supernatant was subjected to BCA protein assay (DQ111-01; TransGen Biotech) to quantify protein levels. For IP, the cell lysates were incubated with the Myc/FLAG magnetic beads (M047-11/M185-11; MBL Life Sciences) for 1 h or overnight at 4°C. The beads were pelleted and washed with lysis buffer three times and heated in 1× denaturing loading buffer for 10 min at 95°C before being resolved by SDS-PAGE. The cell lysates were separated on a 4%–20% Bis-Tris gel (M00656; GenScript), transferred to polyvinylidene difluoride membranes (IPVH00010; Millipore), and probed with antibodies.
Bca protein assay
Manufactured by Transgene
Sourced in China
The BCA protein assay is a colorimetric detection and quantification method used to determine the total protein concentration in a sample. It is based on the reduction of copper ions (Cu2+) to cuprous ions (Cu+) by proteins in an alkaline medium, and the subsequent colorimetric detection of the cuprous ions using a reagent containing bicinchoninic acid (BCA). The resulting purple-colored reaction product exhibits a strong linear absorbance at 562 nm with increasing protein concentrations, allowing for accurate and sensitive protein quantification.
Automatically generated - may contain errors
Lab products found in correlation
Bis tris gel, by GenScript (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ab217330, by Abcam (1 mentions)
Protease inhibitor cocktail, by Transgene (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by MedChemExpress (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Gapdh, by Abways (1 mentions)
Western lightning plus, by PerkinElmer (1 mentions)
4 protocols using bca protein assay
1
Protein Extraction and Immunoprecipitation
2
Western Blot Analysis of TMEM30A
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The TA muscles were lysed in RIPA lysis buffer with protease inhibitor cocktail and ethylenediaminetetraacetic acid (EDTA) (TransGen, China). Protein concentration was determined using a BCA Protein Assay (TransGen, China). Equal amounts of protein were loaded onto a 10% polyacrylamide gel, then separated and transferred to nitrocellulose membranes (Merck Millipore, Germany). Blots were blocked with 8% non-fat dry milk in Tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature and then incubated with primary antibodies in blocking solution overnight at 4 °C. The primary antibodies included TMEM30A (rabbit polyclonal, ab217330, Abcam, USA) and GAPDH (mouse monoclonal, 60004-1-Ig, Proteintech, USA). Primary antibodies were detected with either an anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, USA), and signals were developed using a SuperSignal West Pico Chemiluminescent Substrate (Pierce Protein Biology, USA). ImageJ (v1.8.0) was used to calculate the relative density of the protein.
3
Western Blot Analysis of Protein Expression
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Cell lysates were prepared in RIPA lysis buffer (Beyotime Biotechnology, China) containing PMSF and a protease inhibitor cocktail (MedChemExpress, China), and the protein content of the generated cell lysates was determined using the BCA protein assay (TransGen Biotech, China). Aliquots containing 30 μg of total protein were loaded on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After membranes were blocked with 5% BSA for 1 h, they were probed with indicated primary antibodies overnight at 4 °C, followed by incubation with the HRP-conjugated secondary antibodies (Abcam, 1:5000) for 1 h at room temperature. Primary antibodies were as follows: gD (21719, Santa Cruz, 1:500), DYKDDDDK-Tag (3P8, Abmart, 1:1000), GAPDH (AB0037, Abways, 1:10000), IDO1 (66528, Proteintech, 1:1000).
4
Western Blot Protocol for Protein Detection
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Protein samples were lysed in RIPA buffer with Cocktail (Sangon), quantified with BCA protein assay (Transgene) and separated with 12% SDS-PAGE gel. The separated proteins were transferred to PVDF membrane and then incubated with primary antibodies at 4 °C overnight and secondary antibodies for 1h at room temperature. Films were developed with the Western Lightning PLUS (NEL105001EA, PerkinElmer).
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