Precision red advanced protein assay

Manufactured by Cytoskeleton

Sourced in United States

The Precision Red Advanced Protein Assay is a colorimetric assay designed for the quantification of protein concentrations. It utilizes a proprietary reagent that reacts with proteins, resulting in a color change that can be measured using a spectrophotometer. The assay provides a simple, fast, and accurate method for determining protein levels in a variety of sample types.

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Lab products found in correlation

Chemidoc imaging system, by Bio-Rad (3 mentions) 4xcomplete edta free ultra protease inhibitor cocktail, by Merck Group (2 mentions) 1xphosstop, by Merck Group (2 mentions) Human phospho mapk arrays proteome profiler, by R&D Systems (2 mentions) G lisa activation assay, by Cytoskeleton (2 mentions) Infinite 200 pro nanoquant system, by Tecan (1 mentions) Akta purifier, by GE Healthcare (1 mentions) Superdex 200 column, by GE Healthcare (1 mentions) Luciferase assay reagent, by Promega (1 mentions) Dmem medium, by Thermo Fisher Scientific (1 mentions) Reporter lysis buffer, by Promega (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Glomax 20 20 luminometer, by Promega (1 mentions) Dspe peg 2000 maleimide, by Avanti Polar Lipids (1 mentions) Rhoa g lisa activation assay kit, by Cytoskeleton (1 mentions) Primocin, by InvivoGen (1 mentions) Odyssey blocking buffer, by LI COR (1 mentions) Irdye 680rd, by LI COR (1 mentions) Complete edta free ultra protease inhibitor cocktail, by Merck Group (1 mentions) Phosstop, by Merck Group (1 mentions)

Spelling variants of this product

Precision Red Advanced Protein Assay Reagent (14 citations) Advanced Protein Assay (12 citations) Precision Red (12 citations) Precision Red assay (8 citations) Precision Red Advanced Protein Assay buffer (3 citations)

19 protocols using precision red advanced protein assay

Phospho-MAPK Array Analysis

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Cells were rinsed with cold PBS and immediately lysed in buffer supplemented with 4xcOmplete™ EDTA-free Ultra Protease Inhibitor Cocktail (Sigma-Aldrich) and 1xPhosSTOP™ (Sigma-Aldrich) at 4 °C for 30 min. Following centrifugation at 14,000×g for 5 min, supernatants were transferred into a clean tube and protein concentrations were determined using the Precision Red Advanced Protein Assay (Cytoskeleton, Inc. ADV02-A). Lysates were diluted and analyzed using the Human Phospho-MAPK Arrays (Proteome Profiler; R&D Systems; Minneapolis, MN, USA) according to the manufacturer’s instructions. Nitrocellulose membranes were scanned using a ChemiDoc™ Imaging Systems (BIO-RAD Laboratories, Inc.).

Cruz-Nova P., Schnoor M., Correa-Basurto J., Bello M., Briseño-Diaz P., Rojo-Domínguez A., Ortiz-Mendoza C.M., Guerrero-Aguirre J., García-Vázquez F.J., Hernández-Rivas R., Thompson-Bonilla M.D, & Vargas M. (2018). The small organic molecule C19 binds and strengthens the KRAS4b-PDEδ complex and inhibits growth of colorectal cancer cells in vitro and in vivo. BMC Cancer, 18, 1056.

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Measuring Small GTPase Activity in iPSC-derived MGE Progenitors

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Activity of small GTPases (CDC42, Rac1, and RhoA) in the MGE progenitors derived from the null and direct iPSC lines was measured by G-LISA Activation Assay (Cytoskeleton Inc.) according to the manufacturer's protocol. The MGE progenitors were starved overnight, collected by centrifugation (300 × g, 4 min, RT), plated on 0.03% and/or 0.1% hydrogels, and incubated at 37 °C for 1 h. Afterward, the cells were lysed with an appropriate lysis buffer and centrifugated (10,000 × g, 1 min, 4 °C). Supernatants were immediately frozen and kept at −80 °C until the G-LISA Activity Assay. Protein concentration was measured by Precision Red™ Advanced Protein Assay (Cytoskeleton Inc.).

Szigeti K., Ihnatovych I., Rosas N., Dorn R.P., Notari E., Cortes Gomez E., He M., Maly I., Prasad S., Nimmer E., Heo Y., Fuchsova B., Bennett D.A., Hofmann W.A., Pralle A., Bae Y, & Wang J. (2023). Neuronal actin cytoskeleton gain of function in the human brain. eBioMedicine, 95, 104725.

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RHOA Activation Assay in Jurkat Cells

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We used Jurkat cells expressing wild type HA–RHOA, HA-RHOA
Gly17Val, HA-RHOA Thr19Asn and HA-RHOA Gln63Leu and plated them at
10⁶ cells/ml in RPMI 0.5% FBS. After 24 hours we spun them down
and resuspended in serum-free RPMI 1640 media. For serum stimulation cells were
treated with media containing 10% FBS for 10 min. We washed serum starved and
serum stimulated cells once with ice cold PBS and lysed them in Lysis buffer (50
mM Tris pH 7.5, 10 mM MgCl₂, 0.3 M NaCl and 2% IGEPAL). After
spinning down to remove debris and membranes we quantified lysate protein
content using the Precision Red Advanced Protein Assay (Cytoskeleton, Inc.).
Next we incubated 100 μg of total cleared protein lysate with 20
μl Rhotekin-RBD beads (Cystoskeleton, Inc.) for 1h at 4°C with
rotation. After incubation we washed the Rhotekin-RBD beads with 500 μl
wash buffer (25 mM Tris pH 7.5, 30 mM MgCl₂ and 40 mM NaCl),
resuspended them in 15 μl SDS-PAGE loading buffer. Rhotekin bead samples
were loaded into a Bis-Tris gel and proteins were resolved by electrophoresis in
MES buffer and transferred to a PVDF membrane. The presence of HA-tagged
activated RHOA associated with the Rhotekin-RBD beads was determined by
immunoblotting using an HA antibody following standard procedures.

Palomero T., Couronné L., Khiabanian H., Kim M.Y., Ambesi-Impiombato A., Perez-Garcia A., Carpenter Z., Abate F., Allegretta M., Haydu J.E., Jiang X., Lossos I.S., Nicolas C., Balbin M., Bastard C., Bhagat G., Piris M.A., Campo E., Bernard O., Rabadan R, & Ferrando A. (2014). Recurrent mutations in epigenetic regulators, RHOA and FYN kinase in peripheral T cell lymphomas. Nature genetics, 46(2), 166-170.

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Alcohol-Induced RhoA and Rac1 Activation

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The pool GTP-bound RhoA or Rac1 was measured using the RhoA and Rac1 G-LISA activation assay (Cytoskeleton, Inc., Denver, CO, USA). HUVEC were grown in 10 cm culture dishes to confluence. Medium was changed to EBM for 6 h prior to experiments to alleviate potential confounding effects of serum. The cells were treated with 100 mM alcohol for 1, 5, 10, 30, or 60 min before a wash with ice-cold PBS, and addition of 250 μL ice-cold lysis buffer containing protease inhibitors. The cells were quickly scraped and harvested, clarified by centrifugation at 14000 × g, 4 °C for 2 min, and the supernatants snap-frozen in liquid nitrogen. Protein concentrations were determined with the Precision Red™ Advanced Protein Assay (Cytoskeleton, Inc.) using an Infinite 200 PRO Nanoquant system (Tecan, Männedorf, Switzerland). Protein concentrations ranged from 1.75-2.00 mg/mL and were equalized for the assay. GTP-bound levels of RhoA or Rac1 were determined in 96-well RhoA-GTP or Rac1-GTP binding plates, using a lysis buffer blank negative control and 80 pg/ml recombinant GTP-bound RhoA or Rac1 positive controls. Luminescence was determined with the Infinite 200 PRO Nanoquant system.

Doggett T.M, & Breslin J.W. (2014). Acute Alcohol Intoxication-Induced Microvascular Leakage. Alcoholism, clinical and experimental research, 38(9), 2414-2426.

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Protein Purification via Size-Exclusion

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Protein sample was injected into a Superdex-200 column (GE Healthcare) equilibrated with the running buffer containing 50 mM PBS pH 7.4, 150 mM NaCl by Akta purifier (GE Healthcare). Absorbance at 280 nm was detected. For the method of measuring protein concentration, we used Precision Red Advanced Protein Assay (Cytoskeleton) according to the manufacturer’s protocol.

Zhou X., Zheng Y., Lv Q., Kong D., Ji B., Han X., Zhou D., Sun Z., Zhu L., Liu P., Jiang H, & Jiang Y. (2021). Staphylococcus aureus N-terminus formylated δ-toxin tends to form amyloid fibrils, while the deformylated δ-toxin tends to form functional oligomer complexes. Virulence, 12(1), 1418-1437.

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Hh Inhibition Modulates Foxp3 Expression

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EL4 cells were seeded in triplicate in Treg differentiation medium (20ng/mL IL2, 6ng/mL TGFB, 5μg/mL anti-CD3, and 5μg/mL anti-CD28) for 48 hours. To inhibit Hh activity, 10μM GANT61 (Hh-i) was added for 24 hours after Treg differentiation. Viability was evaluated for all groups and was determined to be greater than 90%. Next, cells were transfected in serum free DMEM medium (Life Technologies) with 200ng pGL3prom 8X Foxp3 plasmid (UAB) and lipofectamine 2000 (Life Technologies) for 12 hours. After transfection, medium was replaced with DMSO or Hh-i (10μM)-containing medium for 4 hours. The cells were then washed in 1X PBS and lysed in 1X reporter lysis buffer (Promega) and placed at −80°C overnight. 20μL of cell lysate was mixed with 100μL Luciferase Assay Reagent (Promega), and luminescence was quantified using a Glomax 20/20 luminometer (Promega) and normalized to total protein concentration determined by using precision red advanced protein assay (Cytoskeleton, Denver, CO). The details for recombinant proteins, antibodies, and reagents are in Supplementary Table S1).

Hinshaw D.C., Benavides G.A., Metge B.J., Swain C.A., Kammerud S.C., Alsheikh H.A., Elhamamsy A., Chen D., Darley-Usmar V., Rathmell J.C., Welner R.S., Samant R.S, & Shevde L.A. (2023). Hedgehog signaling regulates Treg to Th17 conversion through metabolic rewiring in breast cancer. Cancer immunology research, 11(5), 687-702.

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Trastuzumab Conjugation with DSPE-PEG-Maleimide

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Lyophilized Trastuzumab (MedChemExpress) was reconstituted in PBS. First, Trastuzumab was reacted with excess 2-iminothiolane (1:20 mol/mol) in an oxygen free reaction vessel filled with gaseous N2 for one hour. Then, the mixture was filtered using PD 10 desalting columns to remove excess 2-iminothiolane. Trastuzumab concentration was measured using Precision Red Advanced Protein Assay (Cytoskeleton, Inc., Denver, CO) and filtered samples were then incubated stirring overnight with 1,2-distearoyl-sn‑glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)−2000] (ammonium salt) (DSPE-PEG-2000) Maleimide (Avanti Polar Lipids, Birmingham, AL) in a 1:4 Trastuzumab:Lipid mol/mol ratio. Samples were collected after each reaction to measure thiol concentrations using Measure-iT™ Thiol Assay Kit (Sigma Aldrich) following manufacturer's protocol.

Velázquez-Vega L.E., Rivera-Robles M., Sánchez-Álvarez A.O., Vivas-Mejía P.E., Aponte-Reyes M., Cruz-Collazo A.M., Grafals-Ruiz N., Dorta-Estremera S., Hernández-O'Farrill E., Vlaar C.P, & Dharmawardhane S. (2024). Efficacy and delivery strategies of the dual Rac/Cdc42 inhibitor MBQ-167 in HER2 overexpressing breast cancer. Translational Oncology, 44, 101928.

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Protein Purification and Quantification

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Escherichia coli BL21(DE3) strains expressing individual antigens were grown overnight as described [4 (link)]. After purification, proteins were dialyzed into glycerol in Pierce Slide-A-Lyzer dialysis cassettes. Protein concentrations were quantified by using the Precision Red Advanced Protein Assay (Cytoskeleton, Inc.).

Hays M.P., Ericsson A.C., Yang Y, & Hardwidge P.R. (2016). Vaccinating with conserved Escherichia coli antigens does not alter the mouse intestinal microbiome. BMC Research Notes, 9, 401.

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Measuring RhoA Activity in Keratinocytes

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To determine the RhoA activity in control and Par3KO keratinocytes, the RhoA G-LISA^® Activation Assay Kit (BK124-S, Cytoskeleton Inc.) was used. For this, primary keratinocytes were grown for 48 h in FAD/HC medium prior to cell lysis. Protein concentration was determined using the Precision Red Advanced Protein Assay (Cytoskeleton Inc.), and the protein concentration in lysates was adjusted to yield 4.5 mg protein/ml before performing the G-LISA RhoA activity assay. Absorption at 490 nm was measured, and spectrometry values were normalized to total RhoA levels as determined by Western blot analysis.

Dias Gomes M., Letzian S., Saynisch M, & Iden S. (2019). Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity. Nature Communications, 10, 3362.

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Mouse ADRB2 Signaling Assay Protocol

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CINC-1, terbutaline and mouse monoclonal ANTI-FLAG BioM2 antibody (Ab) were purchased from Sigma (St. Louis, MO, USA); ADRB2 polyclonal Ab, FITC conjugated (H-20) was purchased from Bioss (Woburn, MA, USA); GAPDH monoclonal Ab (14C10) was purchased from Cell Signaling; PIK-90 was purchased from Cayman Chemical (Ann Arbor, MI, USA); protein A–Sepharose beads were purchased from Pierce (Rockford, IL, USA); PDE 4D polyclonal Ab (14613) was purchased from Abcam (Cambridge, MA, USA); rolipram was purchased from MP Biomedicals (Waltham, MA, USA); Odyssey Blocking Buffer, IRDye® 680RD and 800CW Donkey anti-Rabbit IgG Abs and IRDye® 680 RD and 800CW Donkey anti-Mouse IgG Abs were purchased from LI-COR (Lincoln, NE, USA); Primocin was purchased from InvivoGen (San Diego, CA, USA); Precision Red Advanced Protein Assay was purchased from Cytoskeleton (Denver, CO, USA); all other reagents were purchased from Fisher Scientific (Waltham, MA, USA).

Rich T.C., Leavesley S.J., Brandon A.P., Evans C.A., Raju S.V, & Wagener B.M. (2021). Phosphodiesterase 4 Mediates Interleukin-8-Induced Heterologous Desensitization of the β2-Adrenergic Receptor. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(10), e21946.

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