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Pro q diamond phosphoprotein stain

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Pro-Q Diamond phosphoprotein stain is a fluorescent stain used to detect phosphorylated proteins in polyacrylamide gels. It is designed to provide a sensitive and specific method for the detection of phosphoproteins.

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Lab products found in correlation

Sypro ruby, by Thermo Fisher Scientific (3 mentions) Chemidoc mp imaging system, by Bio-Rad (3 mentions) Anti flag antibody, by Merck Group (2 mentions) Amersham imager 600rgb, by GE Healthcare (1 mentions) Amersham imager, by Cytiva (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Immobilon p, by Merck Group (1 mentions) Image lab software version 5, by Bio-Rad (1 mentions) F60 sonic dismembrator, by Thermo Fisher Scientific (1 mentions) Typhoon 9400, by GE Healthcare (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions) Peppermintstick, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Pro-Q Diamond Phosphoprotein Gel Stain (53 citations) Pro-Q Diamond (39 citations) Pro-Q diamond stain (12 citations) Pro-Q Diamond Phosphoprotein Blot Stain Kit (2 citations) Pro-Q Diamond phosphoprotein gel stain kit (2 citations)

22 protocols using pro q diamond phosphoprotein stain

1

Phosphoprotein and Total Protein Staining

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2D polyacrylamide gels were stained for 1 h with Pro-Q® Diamond Phosphoprotein Stain (Invitrogen, #P33300). Fixation, washing, and destaining were performed according to the manufacturer’s guidelines. For total protein stain gels were stained overnight with SYPRO® Ruby (Invitrogen, #S12001).
Müller M., Eghbalian R., Boeckel J.N., Frese K.S., Haas J., Kayvanpour E., Sedaghat-Hamedani F., Lackner M.K., Tugrul O.F., Ruppert T., Tappu R., Martins Bordalo D., Kneuer J.M., Piekarek A., Herch S., Schudy S., Keller A., Grammes N., Bischof C., Klinke A., Cardoso-Moreira M., Kaessmann H., Katus H.A., Frey N., Steinmetz L.M, & Meder B. (2022). NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart. Nature Communications, 13, 6209.
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2

SdeA Catalyzes Ub Modifications

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Ub modification reactions were carried out by mixing 1 μM of SdeA-Core (a.a. 211-910) or SdeA-mART (563-910) with 25 μM ubiquitin in a reaction buffer containing 50 mM NaCl and 50 mM Tris pH 7.5, in the presence or absence of 1 mM NAD+. The reactions were incubated for 1h at 37°C and reaction products were assessed by both 8% native PAGE and 12% SDS-PAGE. Native gels were stained with Coomassie, while SDS-PAGE gels were stained with Pro-Q Diamond phosphoprotein stain (Invitrogen) to assay for PDE activity. ADPR-Ub and PR-Ub migrate at the same position on a native gel (labeled as modified Ub), however, only PR-Ub is visible by Pro-Q phosphoprotein stain due to its free phosphoryl group46 (link). Rab33b ubiquitination reactions were performed by the addition of 4 μM of recombinant Flag-Rab33b to the Ub modification reaction described above. The reaction products were analyzed by SDS-PAGE followed by Western blot using an anti-Flag antibody (Sigma-Aldrich) at a 1:2500 dilution. To perform the intracellular PR-ubiquitination assay of Rab33b, plasmids expressing Flag-Rab33b, GFP alone or the indicated GFP-tagged SdeA were co-transfected in NIH HEK293T cells. Whole cell lysates were subjected to immunoprecipitation with Flag beads and the products were analyzed by anti-Flag Western blot. The expression of GFP-SdeA constructs was analyzed by anti-GFP Western blot.
Akturk A., Wasilko D.J., Wu X., Liu Y., Zhang Y., Qiu J., Luo Z.Q., Reiter K.H., Brzovic P.S., Klevit R.E, & Mao Y. (2018). The mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector. Nature, 557(7707), 729-733.
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3

Phosphoprotein Gel Staining Protocol

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Gels after SDS–PAGE were fixed in solution containing 50% (v/v) ethanol and 10% (v/v) acetic acid. On the following day, the gels were washed three times with ddH2O for 10 min and stained with Pro-Q Diamond phosphoprotein stain (Invitrogen) for 90 min. The gels were washed three times with ddH2O for 10 min, destained twice for 30 min in 50 mm sodium acetate, pH 4.0, containing 20% (v/v) acetonitrile and finally washed with ddH2O. Fluorescent bands on the gel were visualized with an Amersham Imager 600RGB (GE Healthcare).
Filatov A., Kruglova N., Meshkova T, & Mazurov D. (2015). Lymphocyte phosphatase-associated phosphoprotein proteoforms analyzed using monoclonal antibodies. Clinical & Translational Immunology, 4(10), e44-.
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4

Phosphorylation Analysis of BAK1 Protein

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Immunoprecipitated full-length BAK1-Flag or recombinant Flag-BAK1 proteins were subjected to SDS-PAGE followed by transfer to PVDF membranes and immunoblot analysis using anti-Flag antibodies (1:5,000 dilution), anti-phosphothreonine antibodies (1:500 dilution), and anti-phosphotyrosine antibodies (1:500 dilution). Immunoblots were scanned using an Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE, USA) for visualization. Blots stained with ProQ Diamond phosphoprotein stain (Invitrogen, Carlsbad, CA, USA) were scanned using a Typhoon Molecular Dynamics phosphor/fluorescence imager.
Oh M.H., Wang X., Kim S.Y., Wu X., Clouse S.D, & Huber S.C. (2014). The Carboxy-terminus of BAK1 regulates kinase activity and is required for normal growth of Arabidopsis. Frontiers in Plant Science, 5, 16.
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5

SdeA Catalyzes Ub Modifications

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Ub modification reactions were carried out by mixing 1 μM of SdeA-Core (a.a. 211-910) or SdeA-mART (563-910) with 25 μM ubiquitin in a reaction buffer containing 50 mM NaCl and 50 mM Tris pH 7.5, in the presence or absence of 1 mM NAD+. The reactions were incubated for 1h at 37°C and reaction products were assessed by both 8% native PAGE and 12% SDS-PAGE. Native gels were stained with Coomassie, while SDS-PAGE gels were stained with Pro-Q Diamond phosphoprotein stain (Invitrogen) to assay for PDE activity. ADPR-Ub and PR-Ub migrate at the same position on a native gel (labeled as modified Ub), however, only PR-Ub is visible by Pro-Q phosphoprotein stain due to its free phosphoryl group46 (link). Rab33b ubiquitination reactions were performed by the addition of 4 μM of recombinant Flag-Rab33b to the Ub modification reaction described above. The reaction products were analyzed by SDS-PAGE followed by Western blot using an anti-Flag antibody (Sigma-Aldrich) at a 1:2500 dilution. To perform the intracellular PR-ubiquitination assay of Rab33b, plasmids expressing Flag-Rab33b, GFP alone or the indicated GFP-tagged SdeA were co-transfected in NIH HEK293T cells. Whole cell lysates were subjected to immunoprecipitation with Flag beads and the products were analyzed by anti-Flag Western blot. The expression of GFP-SdeA constructs was analyzed by anti-GFP Western blot.
Akturk A., Wasilko D.J., Wu X., Liu Y., Zhang Y., Qiu J., Luo Z.Q., Reiter K.H., Brzovic P.S., Klevit R.E, & Mao Y. (2018). The mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector. Nature, 557(7707), 729-733.
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6

Ubiquitination Assay for SdeA-Core

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Ubiquitination reactions were performed by mixing 1 μM WT SdeA-Core or Sde-Core H277A mutant with 12.5 μM K63 linked poly-Ub in a reaction buffer containing 50 mM Tris-HCl pH 7.5 and 50 mM NaCl, in the presence or absence of 1 mM NAD+. The reactions were incubated for 2 hours at 37 °C and reaction products were assessed by 15% SDS-PAGE stained with either Coomassie or Pro-Q Diamond phosphoprotein stain (Invitrogen). Only modified PR-Ub chains are visible by Pro-Q phosphoprotein stain due to its free phosphoryl group 69 (link).
Wan M., Minelli M.E., Zhao Q., Marshall S., Yu H., Smolka M, & Mao Y. (2023). Phosphoribosyl modification of poly-ubiquitin chains at the Legionella-containing vacuole prohibiting autophagy adaptor recognition. Research Square.
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7

Ubiquitin Chain Cleavage Assay

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The cleavage reactions were performed by adding 1 μM of DUB, DupA, or DupB to in vitro synthesized poly-Ub chains or ubiquitinated substrates at 37°C for 2 hours. For poly-Ub chain cleavage, the reaction products were assessed by 15% SDS-PAGE stained with either Coomassie or Pro-Q Diamond phosphoprotein stain (Invitrogen). For the cleavage of ubiquitinated substrates, the supernatants of the cleavage reactions were first collected by centrifugation at 6,000 rpm. The supernatant was concentrated by precipitation with the addition of PPT (0.1% glacial acetic acid, 49.9% ethanol, and 50% acetone) for 1 hour on ice. The protein pellets were resuspended in SDS loading buffer. The resins were washed 3 times with 50 mM Tris pH 8.0 and the remaining proteins attached to the resin were eluted with 1% SDS and 100 mM Tris pH 8.0. Samples were separated by 15% SDS-PAGE followed by Western blot using an anti-Ub antibody or stained with Pro-Q Diamond phosphoprotein stain.
Wan M., Minelli M.E., Zhao Q., Marshall S., Yu H., Smolka M, & Mao Y. (2023). Phosphoribosyl modification of poly-ubiquitin chains at the Legionella-containing vacuole prohibiting autophagy adaptor recognition. Research Square.
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8

Kinase-Catalyzed Biotinylation Assay

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APB (2) was synthesized and characterized by 1H, 13C, and 31P NMR spectroscopy, UV spectroscopy, ESI mass spectroscopy, and MALDI spectrometry, as discussed in the supporting information (Figures S3–S8). For in cell kinase-catalyzed biotinylation, Hela cells were grown in 12 well plates for 2 days in growth media (F-12 containing 10% FBS, 9 units penicillin, and 9 units streptomycin). The growth media was removed, replaced with fresh growth media containing APB (5 mM) or ATP-biotin (5 mM), and incubated at 37°C for 1 hour in a CO2 incubator to allow kinase-catalyed labeling. As a control, staurosporine (1 μM) in fresh growth media was added to the cells for one hour before adding APB. After washing the cells, the reaction mixtures were separated by SDS-PAGE and visualized with SYPRO® Ruby total protein stain. Where indicated, the gel was stained with ProQ Diamond Phosphoprotein stain (Invitrogen), or the proteins were transferred onto a PVDF membrane (Immobilon-P, Milipore) and visualized with streptavidin-Cy5 reagent (Life Technologies) to detect biotinylated proteins. Detailed experimental procedures and data are supplied as supporting information.
Fouda A.E, & Pflum M.K. (2015). A cell permeable ATP analog for kinase-catalyzed biotinylation. Angewandte Chemie (International ed. in English), 54(33), 9618-9621.
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9

Quantitative VDAC Phosphorylation Assay

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Phosphorylation of VDAC was detected using Pro-Q Diamond phospho-protein stain (Invitrogen). The bands were imaged at 532–560 nm excitation on a Fuji gel scanner, and the difference in staining between control and phosphorylated VDAC was quantified using “Multi Gauge V3.0” software. The VDAC band intensities with gel background subtracted were normalized versus intensity of the untreated samples in each corresponding gel. All gels were analyzed in raw, unmodified state.
Zhang Y.X., Zhao W, & Tang Y.J. (2016). Multilevel induction of apoptosis by microtubule-interfering inhibitors 4β-S-aromatic heterocyclic podophyllum derivatives causing multi-fold mitochondrial depolarization and PKA signaling pathways in HeLa cells. Oncotarget, 7(17), 24303-24313.
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10

Phosphoprotein Detection by SDS-PAGE

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Samples containing equal amount of un-phosphorylated and phosphorylated proteins were separated by 12.5% SDS-PAGE and stained by Pro-Q Diamond phosphoprotein stain (Invitrogen, Carlsbad, California, United States) according to the manufacturer instruction. Images were acquired using a BioRAD chemiDoc MP Imaging System (BioRAD). Images were adjusted using Image Lab software Version 5.2 (BioRAD). The same gel was subsequently stained with Coomassie stain.
Singh P., Pesenti M.E., Maffini S., Carmignani S., Hedtfeld M., Petrovic A., Srinivasamani A., Bange T, & Musacchio A. (2021). BUB1 and CENP-U, Primed by CDK1, Are the Main PLK1 Kinetochore Receptors in Mitosis. Molecular Cell, 81(1), 67-87.e9.
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