Protein estimation kit

Manufactured by Bio-Rad

Sourced in United States

The Protein Estimation Kit is a laboratory tool used to quantify the total protein content in a sample. It employs a colorimetric method to measure the protein concentration, providing a reliable and standardized approach for protein determination in various biological and chemical applications.

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Lab products found in correlation

Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions) Acrylamide, by Bio-Rad (2 mentions) Scintillation cocktail, by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Proteinase inhibitor cocktail, by Merck Group (2 mentions) Gentamicin, by Thermo Fisher Scientific (1 mentions) Trypsin edta reagent, by Thermo Fisher Scientific (1 mentions) Anti β actin ab ac 15, by Merck Group (1 mentions) Atp determination kit, by Thermo Fisher Scientific (1 mentions) Hrp conjugated sheep anti mouse igg, by Merck Group (1 mentions) Mouse anti human cd3ζ, by BD (1 mentions) Hybond nitrocellulose membrane, by Cytiva (1 mentions) Ecl western blotting detection kit, by Cytiva (1 mentions) Complete protease inhibitor cocktail tablet, by Roche (1 mentions) Ampicillin, by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions) Trypsin edta 0.25, by Thermo Fisher Scientific (1 mentions) Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions) Ammonium persulfate, by Bio-Rad (1 mentions)

17 protocols using protein estimation kit

Histone Deacetylase Inhibitor Effects on Prostate Cancer Cells

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RPMI 1640 medium, gentamicin and trypsin/EDTA reagents were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Fetal bovine serum (FBS), charcoal/dextran-treated, certified FBS was from Atlanta Biologicals (Lawrenceville, GA, USA). Acrylamide, protein molecular weight standard markers and Protein Estimation Kit were obtained from Bio-Rad (Hercules, CA, USA). The ECL reagent kit was purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). Histone deacetylase inhibitors including sodium butyrate (NaB), valproic acid (VPA), 5α-dihydrotestosterone (DHT) and anti-β-Actin Ab (AC-15) were from Sigma (St Louis, MO, USA). Other HDAC inhibitors, including suberoylanilide hydroxamic acid (SAHA), AR42 and MS-275, were kindly provided by Dr. Ching-Shih Chen at the Ohio State University Comprehensive Cancer Center (Columbus, OH, USA). PxD101 was from Dr. Jue Wang at Medical Oncology section, University of Nebraska Medical Center (Omaha, NE, USA). Rabbit anti-human PAcP Ab (ATM-3) has been described previously [14 (link), 15 (link)]. The respective Abs against androgen receptor (AR) and PSA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Chou Y.W., Lin F.F., Muniyan S., Lin F.C., Chen C.S., Wang J., Huang C.C, & Lin M.F. (2015). Cellular prostatic acid phosphatase (cPAcP) serves as a useful biomarker of histone deacetylase (HDAC) inhibitors in prostate cancer cell growth suppression. Cell & Bioscience, 5, 38.

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Quantifying Cellular Erythromycin Uptake

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Uptake of [¹⁴C]erythromycin was performed according to a previously published procedure [14 (link)] to correlate the gene expression levels to that of functional activity. Briefly, cells were plated in 12-well tissue culture treated plates and treated every day. Upon terminating treatment after 5 days, medium containing drugs was aspirated from wells and cells were washed (3×10 min) with 2 mL of Dulbecco's phosphate-buffered saline (DPBS) containing 130 mM NaCl, 2.5 mM KC1, 7.5 mM Na₂HP₄ 1.5 mM KH₂PO₄, 1 mM CaCl₂ 0.5 mM MgS0₄, 5 mM glucose, and 20 mM HEPES at pH 7.4. Then, 1 mL of DPBS containing [¹⁴C]erythromycin (0.25 μCi/mL) was added to the cells in each well and incubated at 37°C for 30 min. Solutions were removed and uptake was terminated with ice-cold stop solution (200 mM KC1 and 2 mM HEPES). Later, cells were lysed overnight at room temperature with 1 mL of 0.1% (v/v) Triton-X in 0.3 N sodium hydroxide solution. Aliquots (500 μL) of the cell lysate were then added to scintillation vials containing 3 mL of scintillation cocktail (Fisher Scientific) and radioactivity was measured with a scintillation counter (Model LS-6500; BeckmanCounter, Fullerton, CA, USA) with an efficiency of 95% to measure [¹⁴C] compounds. Uptake in each well was normalized to protein content, estimated by the Bio-Rad protein estimation kit using bovine serum albumin (BSA) as the standard.

Kwatra D., Vadlapudi A.D., Vadlapatla R.K., Khurana V., Pal D, & Mitra A.K. (2014). Binary and ternary combinations of anti-HIV protease inhibitors: effect on gene expression and functional activity of CYP3A4 and efflux transporters. Drug metabolism and drug interactions, 29(2), 101-110.

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Immunoblot Analysis of MgrA Protein

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Cell extracts from the post-exponential (OD₆₀₀ = 4) phase of growth were prepared from various staphylococcal strains as described in Supplementary Protocol S1. The concentration of total proteins from clear lysates was determined using the Bio-Rad protein estimation kit and BSA as the standard. Equal amounts of total cellular proteins were separated on 15% polyacrylamide-SDS gels and transferred onto PVDF membranes. Blots were incubated at 20°C with an appropriate dilution (1∶1000) of anti-MgrA-specific monoclonal for 1 h (generous gift from A. Cheung, USA), followed by another 1 h incubation with a 1∶2500 dilution of goat anti-mouse peroxidase (HRP) conjugate. Prestained protein standards were used for molecular mass estimations, and the gel was stained by Coomassie blue to verify that the quantity of proteins was homogenous in each sample. Each experiment was repeated at least three times with different samples.

Romilly C., Lays C., Tomasini A., Caldelari I., Benito Y., Hammann P., Geissmann T., Boisset S., Romby P, & Vandenesch F. (2014). A Non-Coding RNA Promotes Bacterial Persistence and Decreases Virulence by Regulating a Regulator in Staphylococcus aureus. PLoS Pathogens, 10(3), e1003979.

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ATP Quantification in Cell Viability

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The cells were seeded in 96-well plates at a density of 10,000/well and treatment was initiated with 100 μL of the respective PIs and their binary and ternary combinations. Subsequently, upon termination of the treatment after 5 days, medium containing drug solutions was removed and the cells were washed with DPBS (3×10 min). Lysis solution (100 μL) was then added and the lysate was further utilized for the quantitative detection of ATP using ATP Determination Kit (Molecular Probes, Invitrogen) following the manufacturer's instructions. Ouabain (1 mM), sodium azide (1 mM) and 2,4-dinitrophenol (1 mM) served as ATP inhibitors. Luminescence was then measured using a microtiter plate reader (SpectraFluor Plus) and it was normalized to protein content. It was estimated by the Bio-Rad protein estimation kit using BSA as the standard.

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Western Blot Analysis of CD3ζ Protein

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Cells were lysed in 1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate) buffer [phosphate-buffered saline (PBS), 1% CHAPS, 0·5% sodium deoxycholate, 2% sodium dodecyl sulphate] containing complete protease inhibitor cocktail tablets (Roche, Indianapolis, IN, USA) for 30 min, then centrifuged through polymer wool to clear the supernatant. Protein concentrations were established using a protein estimation kit (Bio-Rad, Hercules, CA, USA). Reduced or non-reduced samples containing 20 μg of total protein were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to Hybond nitrocellulose membranes (Amersham Biosciences, Pittsburgh, PA, USA) and probed with mouse anti-human CD3ζ (BD Biosciences) followed by HRP-conjugated sheep anti-mouse IgG (Sigma-Aldrich), both diluted 1:1000 in PBS-Tween/5% skimmed milk powder. Bands were resolved using an ECL Western Blotting Detection kit (Amersham Biosciences), according to the manufacturer's instructions, and the membranes were analysed by exposure to X-ray film.

Bridgeman J.S., Ladell K., Sheard V.E., Miners K., Hawkins R.E., Price D.A, & Gilham D.E. (2014). CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy. Clinical and Experimental Immunology, 175(2), 258-267.

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Cell Culture and Protein Analysis

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RPMI 1640 medium, dimethyl sulfoxide, antibiotic/antimycotic (100×) solution, ampicillin, trypsin–EDTA (0.25%), penicillin–streptomycin–amphotericin, and FBS were purchased from Thermo Fisher Scientific (Waltham, MA, USA). A protein-estimation kit, acrylamide, ammonium persulfate, tetramethylethylene diamine, and prestained SDS-PAGE standard were from Bio-Rad (Hercules, CA, USA) and an enhanced chemiluminescence kit was procured from Thermo Fisher Scientific. Polyvinylidene difluoride membrane was procured from EMD Millipore (Billerica, MA, USA). All antibodies used in this study were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Pei J., Fu B., Jiang L, & Sun T. (2019). Biosynthesis, characterization, and anticancer effect of plant-mediated silver nanoparticles using Coptis chinensis. International Journal of Nanomedicine, 14, 1969-1978.

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Western Blot Detection of Apoptosis Markers

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Following treatment as described above, cells were washed with ice-cold phosphate-buffered saline and lysed in the lysis buffer as described.^{20 (link)} Equal amounts of cell lysates (20 μg each, as determined by Bio-Rad protein estimation kit) were resolved on SDS-PAGE gel and subsequently transferred onto the polyvinylidene difluoride (PVDF) membrane. After blocking in 5% bovine serum albumin for 1 h at room temperature, membranes were washed three times in tris-buffered saline containing Tween 20. The membranes were then incubated overnight at 4 °C with primary antibodies (diluted in 5% bovine serum albumin in tris-buffered saline-Tween 20 buffer) with gentle rocking. After three washes in tris-buffered saline-Tween 20, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Primary antibodies: cleaved PARP, cleaved caspase-3 (Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology). Secondary antibodies: horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG (Cell Signaling Technology). The immunoreactive bands on PVDF membrane were reacted to the ECL detection reagents and visualised by exposure to X-ray films (Kodak, Rochester, NY, USA). Densitometric analysis of the blots was performed by ImageJ software.

Verma N.K., Sadeer A., Kizhakeyil A., Pang J.H., Angela Chiu Q.Y., Tay S.W., Kumar P, & Pullarkat S.A. (2018). Screening of ferrocenyl–phosphines identifies a gold-coordinated derivative as a novel anticancer agent for hematological malignancies. RSC Advances, 8(51), 28960-28968.

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Western blot analysis of protein extracts

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Cells (1 × 10⁶) were seeded in 100 mm plates and allowed to attach overnight and treated with 3 μM/PC at time points described in figures. For whole cell protein, lysates were collected using RIPA buffer containing protease/phosphatase inhibitor cocktail. For nuclear protein, first cytoplasmic extract was collected using buffer A (1 M HEPES pH 7.9, 2 M KCL,o.5 M EDTA pH 8, 0.1 M EGTA pH 7, 0.1 M DTT, 10 % NP40, protease inhibitor) and later nuclear protein extract were collected using buffer B(1 M HEPES pH 7.9, 5 M Nacl,o.5 M EDTA pH 8, 0.1 M EGTA pH 7, 0.1 M DTT and protease inhibitor). The protein concentrations were measured using Bio-Rad protein estimation kit, spectrophotometrically. Equivalent amounts of protein lysates were separated by electrophoresis in 12 % SDS polyacrylamide gel and blotted onto nitrocellulose membrane. The blotted membranes were incubated with different primary antibodies, followed by incubations with secondary antibodies. The proteins were visualized using an ECL kit.

Ravi M., Tentu S., Baskar G., Rohan Prasad S., Raghavan S., Jayaprakash P., Jeyakanthan J., Rayala S.K, & Venkatraman G. (2015). Molecular mechanism of anti-cancer activity of phycocyanin in triple-negative breast cancer cells. BMC Cancer, 15, 768.

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Western Blot Analysis of Signaling Proteins

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The isolated cells (1x10⁶) were lysed with the help of lysis buffer (ThermoFisher USA). The total protein content was analyzed by protein estimation kit (Bio-Rad Lab. USA). About 30 mg of total protein was loaded and separated on SDS-PAGE (10%) and then transferred to nitrocellulose membranes. The membranes were blocked with 0.5% Triton-X-100 in Tris buffered saline along with 5% nonfat milk at 4°C for 12 hours and incubated with primary antibodies of IRS-2 (1:500), PI3K (1:500), VEGF (1:500), Akt (1:500) and actin (1:500).All the antibodies were bought from Santa Cruz Biotech. USA. The membranes were rinsed with tris-buffered saline and incubated along with horseradish peroxidase conjugated II^ry antibody (1:5000) for 90 minutes at 25°C. After incubation, the membranes were rinsed with tris-buffered saline. The densitometric analysis of bands was performed using iBright Western blot imaging system (ThermoFisher USA). The expression of proteins was normalized against Actin which was loading control with the help of Image plus software.

Ji Z., Luo J., Su T., Chen C, & Su Y. (2021). miR-7a Targets Insulin Receptor Substrate-2 Gene and Suppresses Viability and Invasion of Cells in Diabetic Retinopathy Mice via PI3K-Akt-VEGF Pathway. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 14, 719-728.

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Quantitative Immunoblot Analysis of Igf2bp1

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For SDS-PAGE and immunoblotting, DLD1-dnTCF7L2-GFP, DLD1-dnTCF7L2-Igf2bp1, MEF wild-type, and MEF-Igf2bp1-KO cells were harvested, and whole-cell extract (WCE) was prepared by adding 1 mL of cell lysis buffer per 5 × 107 cells. WCE protein content was quantified using a Bio-Rad protein estimation kit. The WCE was subjected to SDS-PAGE (10%) and subsequently transferred to a membrane for immunoblotting. Blots were probed with antibodies against Igf2bp1, Myc, and β-actin obtained from Cell Signaling. Horseradish peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit) were used to detect the respective primary antibodies. Quantitative luminescence of immunoblots was performed using ImageJ software.

Singh V., Walter V., Elcheva I., Imamura Kawasawa Y, & Spiegelman V.S. (2023). Global role of IGF2BP1 in controlling the expression of Wnt/β-catenin-regulated genes in colorectal cancer cells. Frontiers in Cell and Developmental Biology, 11, 1236356.

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