Bca protein assay kit

Manufactured by Tiangen Biotech

Sourced in China

The BCA Protein Assay Kit is a colorimetric detection kit used for the quantification of total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) method to measure the reduction of copper ions by proteins in an alkaline environment, resulting in a purple-colored complex that can be measured spectrophotometrically.

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Lab products found in correlation

Pvdf membrane, by Merck Group (30 mentions) Ripa buffer, by Beyotime (17 mentions) Ab181602, by Abcam (7 mentions) Ripa lysis buffer, by Beyotime (7 mentions) Enhanced chemiluminescence kit, by Cytiva (7 mentions) Protease and phosphatase inhibitor, by Roche (5 mentions) Polyvinylidene difluoride membrane, by Merck Group (5 mentions) Sodium dodecyl sulfonate polyacrylamide gel, by Solarbio (5 mentions) Beyoecl plus, by Beyotime (4 mentions) Ab205719, by Abcam (4 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (4 mentions) Trizol reagent, by Thermo Fisher Scientific (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions) Ecl kit, by Thermo Fisher Scientific (4 mentions) Ab205718, by Abcam (3 mentions) Ab38898, by Abcam (3 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (3 mentions) Gapdh, by Cell Signaling Technology (3 mentions) Anti e cadherin, by Proteintech (3 mentions) Ab16663, by Abcam (3 mentions)

Close spelling variants of this product

BCA protein assay (5 citations) BCA protein kit (2 citations) BCA assay kit (12 citations) BCA assay (3 citations) BCA kit (30 citations)

169 protocols using bca protein assay kit

Protein Expression Analysis in Bovine Muscle

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Approximately 50 mg of frozen BF muscle was taken and homogenized in 200 μL of ice-cold RIPA lysis buffer (Solarbio, Beijing, China) containing 1 mmol/L PMSF, 5 mmol/L NaF and 1 mmol/L Na₃VO₄. The homogenates were shaken on ice for 30 min and then centrifuged (12,000 × g at 4 °C for 15 min) to obtain the supernatant. Ten microliters of supernatant was taken to determine protein concentration using a BCA Protein Assay kit (Tiangen, Beijing, China). The remaining supernatant was diluted to the proper protein concentration with RIPA buffer and then mixed with 5 × loading buffer (CWBIO, Beijing, China) and finally boiled at 100 °C for 10 min to obtain the samples.
The protein expression was measured by Western blot analysis as described previously (Guo et al., 2018 (link)). Beta-tubulin was chosen as the reference protein, and the anti-β-tubulin antibody (1:2,000; catalog number 10094-1-AP) was purchased from Proteintech Group (Rosemont, IL 60018, USA). The antibodies including anti-AMPKα, anti-p-AMPKα, anti-p70S6K, anti-p-p70S6K, anti-eIF2α and anti-p-eIF2a (1:100; catalog numbers 2532, 2531, 9202, 9205, 9722, and 9721, respectively) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies used were goat anti-rabbit or anti-mouse HRP-conjugated (1:4,000; Bioworld Technology).

Liang Z., Jin C., Bai H., Liang G., Su X., Wang D, & Yao J. (2022). Low rumen degradable starch promotes the growth performance of goats by increasing protein synthesis in skeletal muscle via the AMPK-mTOR pathway. Animal Nutrition, 13, 1-8.

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Expression and Purification of Recombinant Enzymes

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Expression and purification of the enzymes were as described previously (Li et al. 2022 (link)). The recombinant cells were incubated in 3 mL LB medium at 37 °C and 220 rpm for 6.0 h. Next, 2 mL of the cultures were transferred to 100 mL of newly prepared LB medium in 500-mL flasks and cultivated at 37 °C. The overexpression of the enzymes was induced by the addition of 0.2 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) when the optical density at 600 nm of the culture reached 0.6, and then the bacteria were incubated at 18 °C and 220 rpm for 16.0 h. After induction, the cells were harvested by centrifugation at 4600×g and 4 °C for 5 min, washed once and resuspended in 20 mM Tris–HCl buffer (pH 8.0). The cells were then lysed by a high-pressure homogenizer supplied by the Shanghai Litu Mechanical Equipment Engineering Co., Ltd. (Shanghai, China). The lysate was centrifuged at 6000×g and 4 °C for 20 min. The protein in the supernatant was purified by Ni-chelating affinity chromatography. The purity of the enzyme was examined by SDS-PAGE (Additional file 1: Figure S1), and the proteins were quantified by the BCA Protein Assay Kit from Tiangen (Beijing, China).

Li Z., Zhong Y., Qing Z, & Li Z. (2024). A circuitous route for in vitro multi-enzyme cascade production of cytidine triphosphate to overcome the thermodynamic bottleneck. Bioresources and Bioprocessing, 11(1), 6.

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Mammary Gland Protein Expression Analysis

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The mammary gland tissues were rapidly separated on ice plate, frozen in liquid nitrogen, and stored at −80°C. Total protein was extracted from tissues of the mammary gland randomly selected from each group and their content was detected by BCA protein assay kit (Beijing Tiangen Biotech Co., Ltd). Total protein (20 μg) per sample was resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore). PVDF membranes were incubated in 5% nonfat dry milk for 2 h. TCTP (1:1000), Mcl-1 (1:1000, Cell Signal Technology, Inc), caspase-3 (1:1000, Cell Signal Technology, Inc), caspase-9 (1:1000, Cell Signal Technology, Inc), Bax (1:1000, Abcam), Bcl-xL (1:200, Abcam), and p53 (1:1000, OriGene Technologies, Inc) antibodies were added and then the corresponding Peroxidase-conjugated affinipure goat anti-rabbit IgG (1:5000, Beijing fir ZhongshanGoldenbridge Biotechnology Corporation) and rabbit anti-mouse IgG (1:5000, Beijing fir ZhongshanGoldenbridge Biotechnology Corporation) secondary antiserum were detected. ECL (Millipore Corporation) was used to detect imprinted membrane (Millipore Corporation). The blots were identified for band densities using Image J 1.45s software.

Zhang J.F., Liu J., Gong G.H., Zhang B, & Wei C.X. (2019). Mongolian Medicine RuXian-I Treatment of Estrogen-Induced Mammary Gland Hyperplasia in Rats Related to TCTP Regulating Apoptosis. Evidence-based Complementary and Alternative Medicine : eCAM, 2019, 1907263.

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Protein Extraction and Western Blot Analysis

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Samples were homogenized in lysis buffer (pH 7.4) containing a protease inhibitor cocktail (HY-K0010, MedChenExpress), phosphatase inhibitor cocktail III (HY-K0023, MedChenExpress), 50 mM Tris, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% NP-40 (Sigma-Aldrich). The protein concentration was determined using a BCA Protein Assay kit (Tiangen Biotech). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and the samples were blotted onto a polyvinylidene fluoride (PVDF) membrane. Antibodies against DPP4 (ab28340, Abcam), F4/80 (28463-1-AP, Proteintech), CD206 (18704-1-AP, Proteintech), p44/42 MAPK (Erk1/2) (137F5) rabbit mAb (4695, Cell Signaling Technology), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, Cell Signaling Technology), signal transducer and activator of transcription 6 (STAT6) (ab32520, Abcam), and Phospho-STAT6 (phospho Y641) (ab263947, Abcam) were used at a dilution of 1:2000. Secondary antibody binding and detection were performed according to standard protocols using an enhanced chemiluminescence (ECL) detection reagent (Bio-Rad).

Zuo B., Li T., Liu X., Wang S., Cheng J., Liu X., Cui W., Shi H, & Ling C. (2023). Dipeptidyl peptidase 4 inhibitor reduces tumor-associated macrophages and enhances anti-PD-L1-mediated tumor suppression in non-small cell lung cancer. Clinical & Translational Oncology, 25(11), 3188-3202.

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Protein Extraction and Western Blot Analysis

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Protein extraction was implemented utilizing RIPA Lysis Buffer (Beyotime). The concentrations of protein samples were examined with a BCA protein assay kit (Tiangen). After separation by 10% sodium dodecyl sulfonate‐polyacrylamide gel (SDS‐PAGE; Solarbio) electrophoresis, the protein sample (20 μg) was blotted onto polyvinylidene difluoride membrane (Corning) and then blocked with 5% non‐fat milk (Solarbio) for 1 h. Afterwards, the membranes were immunoblotted with primary antibodies against CHEK1 (1:10000, ab32531; Abcam), proliferating cell nuclear antigen (PCNA) (1:2000, ab152112; Abcam), Ki67 (1:1000, ab243878; Abcam), matrix metalloproteinase 9 (MMP9) (1:1000, ab38898; Abcam), Bcl‐2‐associated X protein (Bax, 1:1000, ab53154; Abcam) and GAPDH (1:10000, ab181602; Abcam) overnight at 4°C followed by interaction with Goat Anti‐Rabbit IgG H&L (HRP) secondary antibody (1:10000, ab205718; Abcam) for 2 h at indoor temperature. Finally, the intensity of protein bands was assessed utilizing BeyoECL Plus ECL Kit (Beyotime).

Jin M., Zhang F., Li Q., Xu R., Liu Y, & Zhang Y. (2022). Circ_0011292 knockdown mitigates progression and drug resistance in PTX‐resistant non‐small‐cell lung cancer cells by regulating miR‐433‐3p/CHEK1 axis. Thoracic Cancer, 13(9), 1276-1288.

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Recombinant Enzyme Expression and Purification

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The expression of the recombinant enzyme was performed by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the bacterial suspension at the initial concentration of OD600 = 0.7. After 18 h at 16°C, the induced cells were harvested by centrifugation at 4°C for 5 min at 10,000 g and washed twice with PBS buffer (pH 7.4). After sonication and centrifugation at 12,000 g for 10 min, the supernatants was obtained as the crude enzyme solution. To purify the recombinant TtBgl3, the crude enzyme solution was applied onto a Ni-NTA column (Sangon Biotech, China). The TtBgl3 protein was eluted from the column with midazolam buffer by gradient elution. Enzyme fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a 5% (w/v) stacking gel and a 12% (w/v) separating gel. After electrophoresis, SDS-PAGE was stained with Coomassie blue R-250. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Tiangen Biotech, China) (Zhang et al., 2021 (link)).

Qu Y., Luo Y., Yang X., Zhang Y., Yang E., Xu H., He Y., Chagan I, & Yan J. (2022). Highly Efficient Biotransformation of Phenolic Glycosides Using a Recombinant β-Glucosidase From White Rot Fungus Trametes trogii. Frontiers in Microbiology, 13, 762502.

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Recombinant Expression and Purification of OppLTL

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For the expression protein, the ORF of OppLTL was amplified using a specific primer (OppLTL-Fw1/Rv1) containing the restriction enzyme sites BamHI and XhoI. The PCR product was subcloned into the pET-32a (+) vector to construct the expression vector pET-32a-OppLTL. The recombinant plasmid was validated by double enzyme digestion and sequencing. The expression plasmid was transformed into E. coli BL21 (DE3) cells, and the positive clone was screened by PCR and sequencing. Isopropyl β-D-thiogalactopyranoside (IPTG) was used to induce recombinant OppLTL (rOppLTL) at a final concentration of 0.5 mM at 37°C for 8 h. The expression, purification, and refolding of rOppLTL were performed according to the methods of Qu et al. (24 (link)). Meanwhile, recombinant His-tag (rTRX) was expressed and purified as control. The purified proteins were analyzed by 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The concentrations of recombinant proteins were determined by BCA Protein Assay Kit (Tiangen Biotech, China).

Liu J., Liu X., Wang Z, & Zhang Q. (2022). Immunological characterization and function analysis of L-type lectin from spotted knifejaw, Oplegnathus punctatus. Frontiers in Immunology, 13, 993777.

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Protein Extraction and Western Blot

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Cells were lysed with 1 ml RIPA buffer and sonicated on ice. The lysates were quantified using the BCA Protein Assay Kit (TIANGEN Biotech, Beijing, China). The lysates were incubated with 4 ul antibody and 10 ul protein A/G beads (GE Healthcare, Marlborough, MA, USA) overnight at 4 °C, and the beads were washed three times with HTHG buffer and eluted with sodium dodecyl sulfate loading buffer. The proteins were separated and analyzed by western blot with the indicated antibodies.

Zhang P., Wang S., Wang S., Qiao J., Zhang L., Zhang Z, & Chen Z. (2016). Dual function of partitioning-defective 3 in the regulation of YAP phosphorylation and activation. Cell Discovery, 2, 16021-.

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Quantification of Protein Expression via RIPA Lysis and Western Blot

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Protein concentrations, isolated using Radioimmunoprecipitation Assay (RIPA) Lysis Buffer (cat. No. KGP702; KeyGen Biotech), were quantified with BCA Protein Assay Kit (Tiangen Biotech, Beijing, China). Protein samples were separated using 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, then transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% skimmed milk for 3 h at 22°C. The membranes were incubated overnight at 4°C with primary antibodies, including anti-SPARC (1:1000; cat. No. ab207743; Abcam, Cambridge, USA) and anti-GAPDH (1:1000; cat. No. ab9485; Abcam). Next, the membranes were probed with fluorescent rabbit secondary antibodies (cat.
No. 18-4517-32; Rockland Immunochemicals, Rockland, USA) at 22°C for 2 h. Finally, the protein blots were visualized using Odyssey 3.2 (LI-COR Biosciences, Lincoln, USA).

Wen X., Han W, & Liu C. (2023). hsa_circ_0017842 acts as a competing endogenous RNA to enhance the malignancy of gastric cancer. Advances in clinical and experimental medicine : official organ Wroclaw Medical University, 32(7).

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Western Blot Analysis of Nfa34810 Signaling

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RAW264.7 cells were seeded into 6-well plates at a density of 1 × 10⁶ cells/well and stimulated with Nfa34810 for various periods of time. The cells then were lysed with radioimmunoprecipitation assay buffer supplemented with phosphatase and protease inhibitors (CWBIO, China) on ice for 30 min. The supernatant was collected after centrifuging at 12,000 × g at 4°C for 25 min, and then the protein concentration was measured using a BCA protein assay kit (Tiangen Biotechnology, China). Equal protein concentrations were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies, including p-ERK1/2, p-p38, p-JNK, p-p65, p-AKT, NF-κB p-p65, and β-actin. Subsequently, membranes were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Beyotime Biotechnology) or anti-mouse IgG antibodies (SouthernBiotech) and detected using a Western Lightning plus ECL kit (PerkinElmer, USA).

Ji X., Zhang X., Li H., Sun L., Hou X., Song H., Han L., Xu S., Qiu X., Wang X., Zheng N, & Li Z. (2020). Nfa34810 Facilitates Nocardia farcinica Invasion of Host Cells and Stimulates Tumor Necrosis Factor Alpha Secretion through Activation of the NF-κB and Mitogen-Activated Protein Kinase Pathways via Toll-Like Receptor 4. Infection and Immunity, 88(4), e00831-19.

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