Precision plus protein dual color

Manufactured by Bio-Rad

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Precision Plus Protein Dual Color is a pre-stained protein standard that is used for protein molecular weight determination and monitoring protein separation in SDS-PAGE analysis. It contains a mixture of ten recombinant proteins with molecular weights ranging from 10 to 250 kDa, which are labeled with two different colored dyes.

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12 protocols using precision plus protein dual color

Quantifying Cellular Trx2 Levels in T. brucei

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To estimate the cellular level of Trx2, between 0.5 and 5 ng recombinant Trx2 and the lysate of 1 to 3 x 10⁷ PC T. brucei were separated on 14% SDS gels, blotted and probed with the T. brucei Trx2 antibodies as described for Western blot analysis. ImageJ was used to quantify the raw integrated density (the sum of the values of the pixels) of each band. The density of the band corresponding to the highest amount of recombinant Trx2 was set as 100% and the % signal for other protein bands was calculated as a proportion of this. The signals from the two forms of Trx2 detected in the cell lysates were combined to give the total cellular Trx2. The values from seven independent analyses were averaged. To determine the size of the two Trx2 species detected in the cell lysates, the relative mobility of both bands and of recombinant Trx2 was measured and the molecular mass calculated based on standard curves derived from PageRuler Plus (ThermoFisher) and Precision Plus Protein DualColor (BioRad) protein standards.

Currier R.B., Ulrich K., Leroux A.E., Dirdjaja N., Deambrosi M., Bonilla M., Ahmed Y.L., Adrian L., Antelmann H., Jakob U., Comini M.A, & Krauth-Siegel R.L. (2019). An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone. PLoS Pathogens, 15(9), e1008065.

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Immunoblotting Analysis of Orexin Proteins

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Muscle tissues and QM7 cell homogenization, protein extraction and concentration measurement were previously described (Lassiter et al., 2015 (link)). Total proteins (70 μg) for cells and (100 μg) for tissues were assessed by immunobloting using the following polyclonal antibodies: rabbit anti-mouse ORX, rabbit anti-rat ORXR1 and 2 (Interchim, Montlucon, France), and mouse anti-HSP70 (ThermoFisher Scientific, Waltham, MA). After striping, the membrane was re-probed with GAPDH or β-actin as housekeeping proteins (Cell signaling Technology, Danvers, MA). Pre-stained molecular weight marker (precision plus protein Dual color) was used as standard (Biorad, Hercules, CA). The signal was visualized by enhanced chemiluminescence (ECL plus) (GE Healthcare Bio-Sciences, Buckinghamshire, UK) and captured by FlourChem M MultiFlour System (Proteinsimple, Santa Clara, CA). Image Acquisition and Analysis were performed by AlphaView software (Version 3.4.0, 1993–2011, Proteinsimple, Santa Clara, CA).

Nguyen P.H., Greene E., Kong B.W., Bottje W., Anthony N, & Dridi S. (2017). Acute Heat Stress Alters the Expression of Orexin System in Quail Muscle. Frontiers in Physiology, 8, 1079.

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Western Blot Protein Extraction

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Cells were collected and centrifugated at 1300rpm for 5 min. Supernatant was removed and pellet was snap frozen and stored at −20° overnight or lysed immediately. Pellets were lysed in NP-40 lysis buffer (with NaF, Aprotinin, Leupetin and PMSF) vortexed and kept for 30 min at 4° in a rocker. Protein quantification was done using a BCA Protein Assay kit (#23225 Thermo Scientific). WB: 25 μg of whole cell lysate were loaded per lane in Criterion TGX gels 4–20% 26 well (Bio-Rad). Protein ladder: Precision Plus Protein Dual Color (Bio-Rad 161–0374). Gels were run in TGS buffer (#161–0772 Bio-Rad) for 30 min at 200V. Transfer: Using Immobilon-P PVDF membrane (#IPVH00010 Millipore) in TG buffer (#161–0771 Bio-Rad) supplemented with FLASHblot (#R-03090-D50 Advansta) for a faster transfer (25min) at 320mA. PVDF membranes were then let block overnight in Advanblock-Chemi (R-03726-E10).

Diaz-Flores E., Comeaux E.Q., Kim K.L., Melnik E., Beckman K., Davis K.L., Wu K., Akutagawa J., Bridges O., Marino R., Wohlfeil M., Braun B.S., Mullighan C.G, & Loh M.L. (2019). BCL-2 IS A THERAPEUTIC TARGET FOR HYPODIPLOID B-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA. Cancer research, 79(9), 2339-2351.

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SDS-PAGE and Western Blot Protocol

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SDS-PAGE was carried out using Criterion^™ TGX^™ precast 4–15% gradient 18-well midi gels (Bio-Rad, 5671084). Samples were prepared in 4X Laemmli sample buffer (Bio-Rad, 1610747) and 10% β-mercapto ethanol (Acros organics, 149582500). Gels were run in Criterion^™ cell (Bio-Rad, 1656001) using PowerPac^™ power supply (Bio-Rad, 1645050), SDS running buffer (Tris base: 0.2501 M, Glycine: 1.924 M, SDS: 0.03467 M) for 70 minutes at 150 constant volts. Transfer to membranes was carried out in a Criterion^™ Blotter (Bio-Rad, 1704071), transfer buffer (Tris base: 25 mM, Glycine: 192 mM, Methanol: 20% v/v) over 1 hour at 100 constant volts for samples of DAOY Tau-GFP, or 3 hours at 350 constant mA for mHTT MEF. All transfers were carried out at 4°C onto 0.45 μM nitrocellulose membranes (Bio-Rad, 1620167). Membranes were incubated with antibodies overnight at 4°C, washed three times and incubated with secondary Hrp-conjugated antibodies at room temperature for 1 hour. Membranes were then exposed to ECL (Amersham Cytiva, RPN2106) or ECL prime (Amersham Cytiva, RPN2236) prior to imaging using a ChemiDoc^™ touch gel imaging system (Bio-Rad, 1708370). Protein size ladders used were either precision plus protein dual color (Bio-Rad, 161–0374) or Himark pre-stained HMW protein standard (ThermoFisher, LC5699). All images were quantified using Image Lab software (Bio-Rad, version 6.0.1).

Benyair R., Giridharan S.S., Rivero-Ríos P., Hasegawa J., Bristow E., Eskelinen E.L., Shmueli M.D., Fishbain-Yoskovitz V., Merbl Y., Sharkey L.M., Paulson H.L., Hanson P.I., Patnaik S., Al-Ramahi I., Botas J., Marugan J, & Weisman L.S. (2023). Upregulation of the ESCRT pathway and multivesicular bodies accelerates degradation of proteins associated with neurodegeneration. Autophagy reports, 2(1), 2166722.

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Quantification and Analysis of Phosphorylated Proteins

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Total proteins were extracted from chick tissues, quantified, and subjected to Western blot as we previously described [26 (link),27 (link)]. The rabbit polyclonal anti-phospho mechanistic target of rapamycin (mTOR)^Ser2448 (#2971), anti-mTOR (#2972), anti-phospho AMP-activated protein kinase alpha (AMPKα)^Thr172 (#2531), anti-AMPKα1 (#2795), anti-AMPKα2 (#2757) anti-phospho acetyl-CoA carboxylase alpha (ACCα)^Ser79 (#3661), anti-ACCα (#3662), anti-HSP90 (#PA5-17610), and mouse monoclonal anti-HSP70 (#MAI-91159) were used. Protein loading was assessed by immunoblotting with the use of rabbit anti-β actin (#4967) or rabbit anti-vinculin (#V4139). Pre-stained molecular weight marker (Precision Plus Protein Dual Color) was used as a standard (BioRad). All primary antibodies were purchased from Cell Signaling Technology, except for the anti-HSP70 and anti-HSP90 which were purchased from Pierce Thermo Scientific, and anti-vinculin which was purchased from Sigma-Aldrich. The secondary antibodies were used (1:5000) for 1 h at room temperature. The signal was visualized by enhanced chemiluminescence (ECL plus) (GE Healthcare Bio-Sciences) and captured by FluorChem M MultiFluor System (Proteinsimple). Image Acquisition and Analysis were performed by AlphaView software (Version 3.4.0, 1993–2011, Proteinsimple).

Nguyen P., Greene E., Ishola P., Huff G., Donoghue A., Bottje W, & Dridi S. (2015). Chronic Mild Cold Conditioning Modulates the Expression of Hypothalamic Neuropeptide and Intermediary Metabolic-Related Genes and Improves Growth Performances in Young Chicks. PLoS ONE, 10(11), e0142319.

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Hempseed Protein Extraction and Analysis

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Hempseed proteins resulting from sequential extraction were analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Ten micrograms of each protein extract was mixed with Laemmli buffer (2% w/v SDS, 10% glycerol, 5% 2-mercaptoethanol, 62 mM Tris-HCl pH 6.8) and loaded onto 10 × 8 cm vertical 12% polyacrylamide gels. Protein standards (Precision Plus Protein Dual Color, Biorad, Hercules, CA, USA) were used to estimate the molecular weight of proteins. The electrophoretic run was performed at 120 V for 2 h with a Mini Protean System (BioRad, Hercules, CA, USA). Gel staining was performed with Colloidal Coomassie brilliant blue G-250 and the gel image was acquired by a GS-900 densitometer using the software ImageLab (BioRad, Hercules, CA, USA).

Cattaneo C., Givonetti A, & Cavaletto M. (2023). Protein Mass Fingerprinting and Antioxidant Power of Hemp Seeds in Relation to Plant Cultivar and Environment. Plants, 12(4), 782.

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Western Blot Analysis of Phospho-Proteins

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Total proteins were extracted from chicken hypothalamus, quantified, and subjected to Western blot as we previously described (Nguyen et al., 2015 (link)). The rabbit polyclonal anti-phospho mechanistic target of rapamycin (mTOR)^Ser2481 (#2971), anti-mTOR (#2972), anti-phospho AMP-activated protein kinase alpha (AMPKα1/2)^Thr172 (#2531), anti-AMPKα1/2 (#2795), anti-HSP90 (#PA5-17610), goat polyclonal anti-HSP60 (#sc-1052), and mouse monoclonal anti-HSP70 (#MAI-91159) were used. Protein loading was assessed by immunoblotting with the use of rabbit anti-β actin (#4967). Pre-stained molecular weight marker (Precision Plus Protein Dual Color) was used as a standard (BioRad, Hercules, CA). All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA), except for the anti-HSP70 and anti-HSP90 which were purchased from Pierce Thermo Scientific (Rockford, IL) and anti-HSP60 from Santa Cruz Biotechnology (Dallas, TX). The secondary antibodies were used (1:5,000) for 1 h at room temperature. The signal was visualized by enhanced chemiluminescence (ECL plus) (GE Healthcare Bio-Sciences, Buckinghamshire, UK) and captured by FluorChem M MultiFluor System (Proteinsimple, Santa Clara, CA). Image Acquisition and Analysis were performed by AlphaView software (Version 3.4.0, 1993–2011, Proteinsimple, Santa Clara, CA).

Rajaei-Sharifabadi H., Ellestad L., Porter T., Donoghue A., Bottje W.G, & Dridi S. (2017). Noni (Morinda citrifolia) Modulates the Hypothalamic Expression of Stress- and Metabolic-Related Genes in Broilers Exposed to Acute Heat Stress. Frontiers in Genetics, 8, 192.

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Tannic Acid-Venom Interaction Assay

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The ability of tannic acid (50 mg, the same amount used in the in vivo experiments) to interact with venom proteins was assessed using an in vitro simulation in which venom alone (2.4 mg), the supernatant and precipitate of a mixture of venom preincubated with tannic acid, the supernatant of a mixture of venom preincubated with antivenom, antivenom alone, tannic acid alone, and 10 µL of molecular mass markers (Precision Plus Protein ™ Dual Color, Bio-Rad, San Diego, CA, USA) were run on a 10% polyacrylamide gel in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were prepared in 2× standard buffer (65.8 mM Tris-HCl, 26.3% glycerol, 2.1% SDS and 0.01% bromophenol blue) containing 0.5% β-mercaptoethanol (Bio-Rad) at 100 °C for 10 min. The gel was run in a vertical Mini-Protean electrophoresis system (150 V, 30 mA, 15 W, 45 min), stained for 30 min with Coomassie Brilliant Blue G-250 (Bio-Rad) and washed in deionized water, essentially as described elsewhere [8 (link),64 ]. The amount of venom to be used was calculated based on a rat weighing 400 g. Thus, 2.4 mg of venom or 50 mg of tannic acid were each dissolved in 1 mL of phosphate-buffered saline (PBS). The volumes were mixed and incubated for 1 h at 37 °C, followed by centrifugation. Aliquots of 10 µL each were loaded into each well of the gel.

Oliveira I.C., Yoshida E.H., Dini M.M., Paschoal A.B., Cogo J.C., da Cruz-Höfling M.A., Hyslop S, & Oshima-Franco Y. (2021). Evaluation of Protection by Caffeic Acid, Chlorogenic Acid, Quercetin and Tannic Acid against the In Vitro Neurotoxicity and In Vivo Lethality of Crotalus durissus terrificus (South American Rattlesnake) Venom. Toxins, 13(11), 801.

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Western Blot Analysis of Activated T Cells

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Purified stimulated Tconv were lysed in radioimmunoprecipitation assay (RIPA) buffer with Halt protease inhibitor (Thermo Fisher Scientific). Protein concentration was determined by photometry (DU640, Beckman-Coulter). Subsequently, samples were mixed with Laemmli sample buffer containing 2-mercaptoethanol (Bio-Rad Laboratories, Hercules, CA) and boiled for 5 min. The protein samples were loaded onto Mini-PROTEAN TGX^™ 4 to 15% gradient gels (Bio-Rad) and underwent electrophoretic separation. Proteins were then transferred to PolyScreen PVDF Hybridization Transfer Membranes (PerkinElmer, Waltham, MA). Membranes were cut according to the molecular weights of the proteins of interest (as indicated with Precision Plus Protein Dual Color, Bio-Rad) and incubated with primary and horseradish peroxidase (HRP)–conjugated secondary antibodies. We used Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific) and the ChemiDoc^™ imaging system (Bio-Rad), using Image Lab^™ software (Bio-Rad) for image file export. Images were processed with Adobe Photoshop Creative Cloud (auto contrast function).

Quinn WJ I.I.I., Jiao J., TeSlaa T., Stadanlick J., Wang Z., Wang L., Akimova T., Angelin A., Schäfer P.M., Cully M.D., Perry C., Kopinski P.K., Guo L., Blair I.A., Ghanem L.R., Leibowitz M.S., Hancock W.W., Moon E.K., Levine M.H., Eruslanov E.B., Wallace D.C., Baur J.A, & Beier U.H. (2020). Lactate Limits T Cell Proliferation via the NAD(H) Redox State. Cell reports, 33(11), 108500.

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Western Blot Analysis of Activated T Cells

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