Biotinylated nad

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Biotinylated NAD+ is a nicotinamide adenine dinucleotide (NAD+) molecule that has been chemically modified to include a biotin tag. Biotin is a small molecule that can be used to facilitate the detection or purification of biomolecules. The core function of Biotinylated NAD+ is to serve as a labeled substrate for enzymatic reactions or as a tool for affinity-based experiments.

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Lab products found in correlation

Avidin biotin blocking kit, by Vector Laboratories (3 mentions) Anti biotin polyclonal antibody, by Fortis Life Sciences (2 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Phosphatase cocktail, by Roche (1 mentions) Complete tablet, by Roche (1 mentions) Nupage reducing agent, by Thermo Fisher Scientific (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions) Complete protease inhibitor, by Roche (1 mentions) Vectashield, by Vector Laboratories (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Tris buffered saline (tbs), by Bio-Rad (1 mentions) Laemmli buffer, by Bio-Rad (1 mentions) Westernbrighttm ecl, by Advansta (1 mentions) Hrp streptavidin, by Solarbio (1 mentions) Anti his antibody, by Proteintech (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Calf thymus histone h1, by Merck Group (1 mentions) Tris glycine gradient gel, by Bio-Rad (1 mentions)

19 protocols using biotinylated nad

PARP1 E988K Interaction Assay

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U2OS cells were transfected with Flag-PARP1 E988K. Twenty-four hour after transfection cells were lysed in 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40, 10 mM Tris HCl, pH 7.4 supplemented with protease inhibitors cocktail cOmplete tablets (Roche) and Cell Signalling phosphatase cocktail, followed by sonication. Fifty microlitre of lysate was added to a Flag M2 96 well plate (Genscript) and incubated at room temperature for 30 min. Wells were then washed three times in lysis buffer. Totally, 1.2 µg of purified Banf1 protein was then added to relevant wells for 30 min at room temperature. Two microlitre of 250 nM biotinylated NAD⁺ (Trevigen) and activating DNA oligo was added to the wells for 10 min at room temperature, followed by 2 washes in lysis buffer, one wash in PBS-0.1% Triton X-100 and one wash in PBS. Streptavidin-HRP antibody was added (Trevigen 1:500) for 30 min, followed by one wash in PBS-0.1% Triton X-100 and two washes in PBS. Peroxyglo reagent was added to each well and the chemo-illuminescence was read on a plate-reader.

Bolderson E., Burgess J.T., Li J., Gandhi N.S., Boucher D., Croft L.V., Beard S., Plowman J.J., Suraweera A., Adams M.N., Naqi A., Zhang S.D., Sinclair D.A., O’Byrne K.J, & Richard D.J. (2019). Barrier-to-autointegration factor 1 (Banf1) regulates poly [ADP-ribose] polymerase 1 (PARP1) activity following oxidative DNA damage. Nature Communications, 10, 5501.

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PARP Enzyme Activity Assay

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Unfixed cryosections were incubated in an avidin/biotin blocking kit (Vector Laboratories, Burlingame, USA), followed by incubation at 37°C for 2 h in PARP reaction mixture containing 10 mM MgCl₂, 1 mM DTT, 5 µM biotinylated NAD⁺ (Trevigen, Gaithersburg, USA) in 100 mM Tris buffer with 0.2% Triton X-100 (pH 8.0). Biotin incorporation was detected by avidin - Alexa Fluor 488 conjugate (1∶800, 1 h at room temperature). For controls biotinylated NAD+ was omitted from the reaction mixture [14] (link).

Arango-Gonzalez B., Trifunović D., Sahaboglu A., Kranz K., Michalakis S., Farinelli P., Koch S., Koch F., Cottet S., Janssen-Bienhold U., Dedek K., Biel M., Zrenner E., Euler T., Ekström P., Ueffing M, & Paquet-Durand F. (2014). Identification of a Common Non-Apoptotic Cell Death Mechanism in Hereditary Retinal Degeneration. PLoS ONE, 9(11), e112142.

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TNKS and PTEN ADP-Ribosylation Assays

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Samples containing 0.2 μg of recombinant baculovirus-derived TNKS1 or TNKS2 (purchased from Sigma-Aldrich) and 2 μg of recombinant Escherichia coli-derived MBP-PTEN, MBP-PTEN-AA, or MBP-PTEN-3A were incubated in 50 μL of PARP reaction buffer (50 mM Tris-HCl at pH 8.0, 4 mM MgCl₂, 0.2 mM dithiothreitol) with or without 25 μM biotinylated NAD⁺ (Trevigen) for 30 min at 25°C. Reactions were terminated by addition of 2× sample buffer and analyzed by immunoblotting using an anti-biotin HRP antibody.
For the in vitro ribosylation assay of PARP1, samples containing 0.2 μg of recombinant PARP1 (purchased from Trevigen) and 2 μg of recombinant E. coli-derived GST-PTEN were incubated in 50 μL of PARP reaction buffer (20 mM Tris-HCl at pH 7.5, 50 mM NaCl, 7.5 mM MgCl₂, 0.2 mM TCEP) with 1 μM dsDNA (5′-GAGTGTTGCATTCCTCTCTGGGCGCCGGGCAGGTACCTGCTG-3′) and 5 mM NAD⁺ for 30 min at room temperature. The reactions were stopped by the addition of SDS loading buffer containing 0.1 M EDTA, and samples were analyzed by immunoblotting using an anti-PAR antibody.

Li N., Zhang Y., Han X., Liang K., Wang J., Feng L., Wang W., Songyang Z., Lin C., Yang L., Yu Y, & Chen J. (2015). Poly-ADP ribosylation of PTEN by tankyrases promotes PTEN degradation and tumor growth. Genes & Development, 29(2), 157-170.

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Evaluating Protein deMARylation Activity

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To perform deMARylation assay, reaction mixture containing 10 μM HsPARP10 CatD and 100 μM biotinylated NAD⁺ (Trevigen) in reaction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.5 mM dithiothreitol) was incubated at room temperature for 30 min. The reaction mixture was dialyzed against reaction buffer to remove residual NAD⁺. Five micromoles of yeast Poa1p (ScPoa1p) or various concentrations of DrPARG (2.5–10 μM) was added into the reaction, followed by incubation at room temperature for another 30 min. The reaction was terminated by adding 1% SDS and proteins were resolved in 15% acrylamide gel. The gel was transferred onto a PVDF membrane (Bio-Rad) and probed with anti-biotin polyclonal antibody (Bethyl Laboratories Inc.) (1/5000).

Cho C.C., Chien C.Y., Chiu Y.C., Lin M.H, & Hsu C.H. (2019). Structural and biochemical evidence supporting poly ADP-ribosylation in the bacterium Deinococcus radiodurans. Nature Communications, 10, 1491.

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Profiling PARP-Mediated Protein PARylation

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HeLa cells growing in plates were washed twice with PBS and then incubated with PARP reaction buffer (56 mM HEPES, pH = 8.0, 28 mM KCl, 2mM MgCl2, 0.01% digitonin, 12.5 μM biotinylated-NAD (Trevigen), supplemented with 100 nM Olaparib or 0.01% DMSO) for 30 minutes at 37^oC. Cells were then collected and lysed in pre-heated denaturing lysis buffer [400 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl pH 7.5, 0.5% NP-40, 70 mM ß-Mercaptoethanol, 1.5 mM MgCl2, 10% Glycerol, 5 mM NEM, 1 μM ADP-HPD (a PARG inhibitor) and protease inhibitors] and heated for 10 min at 95 °C. After incubating for 30 min at 4 ^oC, lysates were cleared by centrifugation and then incubated with Streptavidin M-280 Dynal beads (Invitrogen) to trap Biotin- containing proteins according to manufacturer’s guidelines.

Hu Y., Petit S.A., Ficarro S.B., Toomire K.J., Xie A., Lim E., Cao S.A., Park E., Eck M.J., Scully R., Brown M., Marto J.A, & Livingston D.M. (2014). PARP1-driven Poly-ADP-ribosylation Regulates BRCA1 Function in Homologous Recombination Mediated DNA Repair. Cancer discovery, 4(12), 1430-1447.

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Tankyrase-AMOT Protein Interaction Assay

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Recombinant baculovirus-derived GST-TNKS1 (amino acids 1000-1328) (0.2 μg, Sigma #SRP0422) and HEK293T-purified SFB-AMOT proteins were incubated in 30 μL reaction buffer (50 mM Tris-HCl at pH 8.0, 4 mM MgCl2, 0.2 mM dithiothreitol) with or without 25 mM biotinylated NAD⁺ (Trevigen) at 25°C for 30 min. Reactions were terminated and subjected to Western blotting.

Wang W., Li N., Li X., Tran M.K., Han X, & Chen J. (2015). Tankyrase inhibitors target YAP by stabilizing angiomotin family proteins. Cell reports, 13(3), 524-532.

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Cell-free ADP-ribosylation Assay Protocol

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For cell-free ADP-ribosylation assay, 50 μg of whole-cell lysate were used. ADP-ribosylation reaction was performed for 30 min at 37°C with 1 μg NarE or NarE-R7K in a buffer containing 20 mM Tris-HCl (pH 8), 1 mM EDTA, 1 mM DTT, 5 mM MgCl₂, 10 μM biotinylated NAD⁺ (Trevigen) and Complete protease inhibitor (Roche, Basel, Switzerland) (according to the manufacturer’s manual). The reaction was stopped by adding 4x NuPAGE LDS Sample Buffer (Invitrogen) and 10x NuPAGE Reducing Agent (Invitrogen) and boiling the samples for 5 min at 100°C. The samples were subjected to SDS-PAGE and subsequently the biotin-ADP-ribosylated proteins were transferred to a nitrocellulose membrane and visualized with HRP-conjugated streptavidin.

Valeri M., Zurli V., Ayala I., Colanzi A., Lapazio L., Corda D., Soriani M., Pizza M, & Rossi Paccani S. (2015). The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity. PLoS ONE, 10(5), e0127614.

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PARP Activity in Mouse Eye Tissue

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Eyes from wt and rd2 mice were enucleated, frozen immediately on dry ice (-72°C), followed by cryosectioning. A biotin-avidin blocking kit (Vector) was used to block endogenous biotin and to reduce background. After incubation with PARP reaction mixture (10 mM MgCl₂, 1 mM dithiothreitol, 5 μM biotinylated NAD (Trevigen, Gaithersburg, MD, USA) in 100 mM Tris buffer with 0.2% Triton X100, pH 8.0) for 2.5 h at 37°C, the sections were washed 3 times with PBS (5 min). The biotin incorporated by PARP activity was then detected by fluorescently labelled avidin (1:800 in PBS, 1h at RT). After three washes in PBS (5 min), the sections were mounted in Vectashield (Vector). For controls, biotinylated-NAD⁺ was omitted from the reaction mixture resulting in absence of detectable reaction product.

Sahaboglu A., Sharif A., Feng L., Secer E., Zrenner E, & Paquet-Durand F. (2017). Temporal progression of PARP activity in the Prph2 mutant rd2 mouse: Neuroprotective effects of the PARP inhibitor PJ34. PLoS ONE, 12(7), e0181374.

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PARP Activity Assay in Pde6a Mutants

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Unfixed cryosections from Pde6a mutants were incubated with an Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, CA, USA), followed by incubation at 37 °C for 2 h in PARP reaction mixture containing 10 mM MgCl₂, 1 mM DTT, 5 mM biotinylated NAD⁺ (Trevigen, Gaithersburg, MD, USA) in 100 mM Tris buffer with 0.2% Triton X-100 (pH 8.0). Incorporated biotin was detected by binding to avidin conjugated with Alexa Fluor 488 (1:800, 1 h at room temperature; Life Technologies, Darmstadt, Germany). For controls, biotinylated NAD⁺ was omitted from the reaction mixture (not shown, but see Paquet-Durand et al.^{10 (link)}).

Jiao K., Sahaboglu A., Zrenner E., Ueffing M., Ekström P.A, & Paquet-Durand F. (2016). Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations. Cell Death Discovery, 2, 16040-.

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Macrodomains Hydrolysis Activity Assay

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Proximal ADP-ribose hydrolysis activity of the macrodomains was tested with immunoblotting. PARP10 (0.5 μM) and SRPK2 (2 μM) were incubated in 50 mM Tris pH 7.0 with or without 1 μM biotinylated NAD⁺ (Trevigen, USA) at 22 °C for three hours. Then 1 μM of TbMDO or TcMDO was added to the reaction and the incubation was continued for another three hours. The reactions were stopped by adding 2× Laemmli buffer (Bio-Rad, USA) and heating the mixture at 95 °C for 5 min. The samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Whatman, UK). The membrane was blocked using 1% Casein in 1xTBS (Bio-Rad, USA) and visualized with 1:15000 streptavidin conjugated horseradish peroxidase (PerkinElmer, USA) and chemiluminescent substrate (WesternBright^TM ECL, Advansta Corporation, USA).

Haikarainen T, & Lehtiö L. (2016). Proximal ADP-ribose Hydrolysis in Trypanosomatids is Catalyzed by a Macrodomain. Scientific Reports, 6, 24213.

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