Bca quantification kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Australia

The BCA quantification kit is a laboratory reagent used for the quantification of total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) method to produce a colorimetric reaction, allowing for the measurement of protein levels using a spectrophotometer. The kit provides the necessary reagents and protocols to perform this analysis.

Automatically generated - may contain errors

Lab products found in correlation

Pvdf membrane, by Merck Group (4 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions) Chemidoc xrs system, by Bio-Rad (3 mentions) Pierce ecl plus western blot substrate, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Mini gel system, by Bio-Rad (1 mentions) Akt isoform antibody sampler kit, by Cell Signaling Technology (1 mentions) Pierce ecl western blot substrate, by Thermo Fisher Scientific (1 mentions) Gel doc xr system, by Bio-Rad (1 mentions) Anti gapdh, by Abcam (1 mentions) Synergy 2, by Agilent Technologies (1 mentions) Dna extraction kit, by Takara Bio (1 mentions) Primestar, by Takara Bio (1 mentions) Dna free kit, by Thermo Fisher Scientific (1 mentions) Qpcr mix, by Toyobo (1 mentions) Cfx384 real time system, by Bio-Rad (1 mentions) Goscript reverse transcription system, by Promega (1 mentions) Ripa buffer, by Beyotime (1 mentions)

11 protocols using bca quantification kit

Protein Expression and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

R1 cells were grown in 6-well plates seeded with CD1 MEFs as feeders, and treated by lentiviruses as described in the Apoptosis and cell cycle assays section. Total cellular proteins were then extracted using RIPA buffer (Thermo Fisher Scientific) with 1× proteinase and phosphatase inhibitors (Thermo Fisher Scientific). Proteins were quantified with a BCA-Quantification kit (Thermo Fisher Scientific), and subjected to 10% SDS-PAGE gel electrophoresis using BioRad mini-gel system and subsequently transferred to PVDF membranes.
The blotted membranes were then blocked with 5% non-fat dry milk in TBS-T and incubated with primary antibodies at 4°C overnight. The antibodies used were as follows: anti-GAPDH (1/2500, Abcam). Antibodies against p53, Mdm2, pMdm2, and pGSK3β (1/1000 for all) were from Cell Signaling. All Akt isoforms, phospho-Akt Ser473, and pan-Akt were detected using Akt isoform antibody sampler kit from Cell Signaling. Membranes were then washed and blotted with HRP conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Chemiluminescence was detected either using Pierce ECL Western-Blot Substrate (Thermo Fisher Scientific) and X-ray film exposure, and quantified using the ImageJ software (Schneider et al., 2012 (link)), or detected and quantified using BioRad Gel Doc XR System (Hercules, CA, USA).

Wang L., Huang D., Jiang Z., Luo Y., Norris C., Zhang M., Tian X, & Tang Y. (2017). Akt3 is responsible for the survival and proliferation of embryonic stem cells. Biology Open, 6(6), 850-861.

+ Open protocol

+ Expand

Inflammatory Mediators Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols

The concentration of inflammatory mediators (IL-6, IL-1β and TNF-α) in hippocampal homogenate and serum obtained 24 h after surgery, or supernatant of cell culture medium collected after stimulating with LPS for 24 h was measured by enzyme-linked immunosorbent assay according to the manufacturer's instructions (Invitrogen, cat: 88-7324-88, 88-7064-88, 88-7013-88, USA). The results were read at 450 nm with a spectrophotometer (Synergy2, BioTek, USA). Hippocampal protein concentrations were quantified using a BCA quantification kit (Thermo, cat: 23228, 23224, Rockford, IL, USA).

Li H.R., Liu Q., Zhu C.L., Sun X.Y., Sun C.Y., Yu C.M., Li P., Deng X.M, & Wang J.F. (2023). β-Nicotinamide mononucleotide activates NAD+/SIRT1 pathway and attenuates inflammatory and oxidative responses in the hippocampus regions of septic mice. Redox Biology, 63, 102745.

+ Open protocol

+ Expand

Purification of Nanobodies from E. coli

Check if the same lab product or an alternative is used in the 5 most similar protocols

The plasmid containing the nanobody gene of the positive clone in ELISA was transformed into E. coli WK6 competent cells. The clone was grown in Terrific Broth [56 ] supplemented with ampicillin, until the absorbance at 600 nm reached 0.9. Nanobody expression was then induced with 1 mm isopropyl dthiogalactopyranoside for 16 h at 28 °C. After pelleting cells, the periplasmic proteins were extracted by osmotic shock [57 (link)]. This periplasmic extract was loaded on a HisTrap HP 5 mL (Amersham Bioscience, Uppsala, Sweden) column in FPLC. After washing, the histidine tagged proteins were eluted with 250 mm imidazole. The eluted fraction was dialyzed against PBS and the purity was checked by SDS/PAGE and the final yield was determined by BCA quantification kit (Thermo Scientific™, Rockford, IL, USA).

Hmila I., Vaikath N.N., Majbour N.K., Erskine D., Sudhakaran I.P., Gupta V., Ghanem S.S., Islam Z., Emara M.M., Abdesselem H.B., Kolatkar P.R., Achappa D.K., Vinardell T, & El‐Agnaf O.M. (2022). Novel engineered nanobodies specific for N‐terminal region of alpha‐synuclein recognize Lewy‐body pathology and inhibit in‐vitro seeded aggregation and toxicity. The Febs Journal, 289(15), 4657-4673.

+ Open protocol

+ Expand

HO-1 Expression in EAhy926 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

EAhy926 cells were incubated with serum-free DMEM media (blank), C, R, or CR at 50 μg/mL in T75 cell flasks for 24 hours. The cells were then lysed with RIPA lysis buffer (Thermo Fisher Scientific, Australia) and the protein was collected and quantified using BCA quantification kit (Thermo Fisher Scientific, Australia). Equal amounts of proteins were then separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked by 5% milk and then incubated overnight at 4°C with rabbit polyclonal antibodies against heme oxygenase-1 (HO-1, 1 : 1000, Cell Signaling Technology, USA) and beta-actin (1 : 1000, Cell Signaling Technology, USA), followed by anti-rabbit horseradish peroxidase-conjugated secondary antibodies. Then, the membranes were exposed to Pierce ECL Plus western blot substrate (Thermo Fisher Scientific, Australia). The band intensities of the membranes were quantified by ImageJ, and the control for equivalent protein loading was assessed by anti-β-actin antibody.

Zhou X., Afzal S., Zheng Y.F., Münch G, & Li C.G. (2021). Synergistic Protective Effect of Curcumin and Resveratrol against Oxidative Stress in Endothelial EAhy926 Cells. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 2661025.

+ Open protocol

+ Expand

Quantitative Protein Analysis of ADHLSCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total protein lysates were obtained by dissolving ADHLSC pellets in RIPA buffer [50 mM Tris base, pH 8.0, 150 mM NaCl, 1% Triton X-100 0.5% sodium deoxycholate, 0.1% SDS, with protein inhibitors cocktail without EDTA (Roche)]. Protein samples were sonicated shortly and incubated for 30 minutes at 4°C prior to sample clarification by centrifugation (15 min maximum speed). Subsequently, sample supernatants were collected and a total protein quantification was performed (BCA quantification kit, Thermofisher). Twenty μg of total protein extracts were dissolved in loading buffer [Tris-HCl (pH 6.8), glycerol, SDS, DTT, and bromophenol blue], denatured at 95°C for 5 minutes and loaded on a 10% Tris-glycine SDS-PAGE gel for protein separation and transferred overnight at 4°C onto PVDF membranes. Membranes were incubated with 5% BSA blocking solution for 90 min at room temperature. CD200 antibody [0.1 μg/ml] was incubated 90 min at room temperature, and the membranes were thoroughly washed 3× with PBS-T, incubated with fluorescently labelled secondary antibody (Biotium) for 40 min at room temperature, and detected by Li-cor scanner (Odyssey). Quantification analysis was performed by Image Studio Lite Software (Odyssey).

El-Kehdy H., Sargiacomo C., Fayyad-Kazan M., Fayyad-Kazan H., Lombard C., Lagneaux L., Sokal E., Najar M, & Najimi M. (2017). Immunoprofiling of Adult-Derived Human Liver Stem/Progenitor Cells: Impact of Hepatogenic Differentiation and Inflammation. Stem Cells International, 2017, 2679518.

+ Open protocol

+ Expand

Western Blot, RT-qPCR, and Genomic PCR Protocols

Check if the same lab product or an alternative is used in the 5 most similar protocols

For western blot, cells were lysed in RIPA buffer with protease inhibitor cocktail (Roche). Protein lysates were quantified using a BCA quantification kit (Thermo Fisher Scientific), subjected to SDS-PAGE and electrotransferred to PVDF membranes (Millipore). Then the membranes were blocked with 5% milk, and incubated with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. The results of western blotting were obtained by ChemiDoc XRS system (Bio-Rad). For RT-qPCR, total RNA was extracted in TRIzol (Thermo Fisher Scientific) and genomic DNA was removed using a DNA-free kit (Thermo Fisher Scientific). cDNA was generated by the GoScript Reverse Transcription System (Promega). RT-qPCR was performed using the qPCR Mix (TOYOBO) in a CFX384 Real-Time system (Bio-Rad). For genomic PCR, a DNA extraction kit (TIANGEN) was used to extract genomic DNA and PCR was carried out with PrimeSTAR (TAKARA).

Deng L., Ren R., Liu Z., Song M., Li J., Wu Z., Ren X., Fu L., Li W., Zhang W., Guillen P., Izpisua Belmonte J.C., Chan P., Qu J, & Liu G.H. (2019). Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis. Nature Communications, 10, 3329.

+ Open protocol

+ Expand

Quantifying Protein Levels by Western Blotting

Check if the same lab product or an alternative is used in the 5 most similar protocols

Western blotting was performed to determine the protein levels as per the method detailed described in a previous study [28 ]. Cells were lysed with RIPA buffer (Beyotime, China). Then, the supernatant was collected after centrifugation (4℃, 10 min) and denatured at 95 °C for 5 min before cooling on ice. Following quantification using the BCA quantification kit (Thermo Fisher Scientific, USA). Samples were separated according to different molecular weights using 10% SDS-PAGE. A PVDF membrane (Millipore, USA) was immersed in 5% skimmed milk as a blocking buffer for 1 h after transferred. Then it incubated with primary antibodies at 4℃ overnight, therewith secondary antibodies. The PVDF membrane was then developed and photographed. Antibodies are shown in the table S3 below.

Liu T.Y., Hu C.C., Han C.Y., Mao S.Y., Zhang W.X., Xu Y.M., Sun Y.J., Jiang D.B., Zhang X.Y., Zhang J.X., Wang J., Qiao X.P., Pan J.Y., Yang S.Y, & Yang K. (2023). IGF2BP2 promotes colorectal cancer progression by upregulating the expression of TFRC and enhancing iron metabolism. Biology Direct, 18, 19.

+ Open protocol

+ Expand

Western Blotting Protocol with RIPA Lysis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For western blotting, as previously reported (29 (link)), cells were lysed in RIPA buffer with the protease inhibitor cocktail (Roche). Protein lysates were quantified with a BCA quantification kit (Thermo Fisher Scientific), subjected to SDS-PAGE and electrotransferred to PVDF membranes (Millipore). Membranes were then blocked with 5% non-fat milk in TBST (20 mM Tris–HCl, pH 7.5, 140 mM NaCl, 0.1% Tween-20), incubated with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. Western blotting results were obtained using ChemiDoc XRS system (Bio-Rad), and quantification was performed with ImageJ.

Wu Z., Shi Y., Lu M., Song M., Yu Z., Wang J., Wang S., Ren J., Yang Y.G., Liu G.H., Zhang W., Ci W, & Qu J. (2020). METTL3 counteracts premature aging via m6A-dependent stabilization of MIS12 mRNA. Nucleic Acids Research, 48(19), 11083-11096.

+ Open protocol

+ Expand

Evaluating HO-1 Expression in RAW 264.7 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

RAW 264.7 cells were incubated with media, G, T or G-T (5:2, w/w) at 12.5 µg/mL in T75 cell flasks for 24 h before the stimulation of LPS (1 µg/mL). The cells were then lysed with RIPA lysis buffer (Thermo Fisher Scientific, Australia) and the protein was collected and quantified using a BCA quantification kit (Thermo Fisher Scientific, Australia). An equal amount of protein from each sample was then separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked by 5% milk, then incubated overnight at 4 °C with rabbit polyclonal antibodies against HO-1 (1:1000) (cell signalling technologies, Danvers, MA, USA), or β-actin (1:1000) (Cell Signalling Technologies, USA), followed by horseradish peroxidase-conjugated secondary antibodies. Then the membranes were exposed with Pierce ECL Plus Western blot substrate (Thermo Fisher Scientific, Australia). The band intensities of the membranes were quantified by ImageJ, and the control for equivalent protein loading was assessed by anti-β-actin antibody.

Zhou X., Afzal S., Wohlmuth H., Münch G., Leach D., Low M, & Li C.G. (2022). Synergistic Anti-Inflammatory Activity of Ginger and Turmeric Extracts in Inhibiting Lipopolysaccharide and Interferon-γ-Induced Proinflammatory Mediators. Molecules, 27(12), 3877.

+ Open protocol

+ Expand

Quantification of Inflammatory Cytokines in Mouse Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols

Tissue samples from mice were homogenized in lysis buffer (50 mM Tris-HCl buffer pH 8.0 with 120 mM NaCl, 1 mM EDTA, 6 mM EGTA, 1% NP-40 and 1 mM dithiothreitol) supplemented with phosphatase inhibitors and protease inhibitor (Sangon Biotech, China). The lysis product was centrifuged at 12000g for 3 min, and the supernatant was taken for protein quantification according to the manufacturer’s instructions of the BCA quantification kit (Thermo Fisher Scientific, USA), and all the sample protein concentrations were adjusted to 2 mg/ml with lysis buffer. Mouse IL-1β, IL-6, TNF-a and TGF-β1 levels in the extracts were measured according to the manufacturer’s instructions, using specific ELISA kits (MultiSciences Biotech, China) respectively. Three replicate wells were set up for each sample and the results were expressed in pg/mg.

Wang Z., Xiong B., Kang N., Pan X., Wang C., Su L., Xing Z, & Hong J. (2021). The Value of MR-DWI and T1 Mapping in Indicating Radiation-Induced Soft Tissue Injury. Frontiers in Oncology, 11, 651637.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!