Dcp bio1

Manufactured by Kerafast

Sourced in United States

The DCP-Bio1 is a laboratory instrument designed for the detection and analysis of biological samples. It utilizes advanced technology to provide accurate and reliable results. The core function of the DCP-Bio1 is to perform sensitive and precise measurements on a variety of biological materials.

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DCP-Bio1 probe (2 citations)

11 protocols using dcp bio1

PTEN-AIF Interaction and Modification Analysis

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Recombinant full-length PTEN, AIF, and its mutants were expressed in E. coli using the SUMO-fusion expression system (Life Sensor, Tokyo, JP) and purified by nickel chelating, ion exchange, and gel filtration columns as reported 67 (link). Recombinant proteins were kept in 50 mM Tris–HCl, pH 7.8. Incubation reaction was carried out by incubating PTEN and AIF alone or together in the presence of 200 μM NADH (Sigma-Aldrich) and protease inhibitors PMSF and cocktail at 4°C for 1 h. Afterward, the mixtures were prepared under reducing or non-reducing condition and analyzed with Western blots. For DCP-Bio1 (KeraFAST, Boston, MA) labeling, the mixtures were incubated with 500 μM DCP-Bio1 in the presence of 50 mM IAA at 4°C for additionally 1 h. Finally, the mixtures were prepared under reducing condition and analyzed with Western blots.

Shen S.M., Guo M., Xiong Z., Yu Y., Zhao X.Y., Zhang F.F, & Chen G.Q. (2015). AIF inhibits tumor metastasis by protecting PTEN from oxidation. EMBO Reports, 16(11), 1563-1580.

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Detecting IL-17A Induced Oxidative Stress in Cells

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Cells were switched to serum-free medium for 2 hours; in the final hour, cells were preincubated with 1 mM DCP-Bio1 (Kerafast, Boston, MA) in serum-free medium for 1h at 37° C. Medium containing DCP-Bio1 was removed, and fresh serum-free medium containing 100 ng/ml IL-17A was added to cells for indicated times. Cells were then fixed in 4% formalin and permeabilized with 0.2% Triton X100 in PBS for 10 min. The permeabilized cells were blocked in 5% BSA in PBS for 1h. The cells were then incubated with fluorescently labeled streptavidin (1:2000, SA alexa fluor 647) and the nucleus was counterstained with DAPI (1:4000). Cell images were acquired using a Zeiss LSM 510 META Confocal Laser Scanning Imaging System. All images were taken at 20× magnification. The image files were converted to tiff format and brightness and contrast were adjusted equally in all images.
Alternatively, cells were harvested in the presence of dimedone to detect oxidized cysteines in cell lysates according to a previously described method [37 ]. In brief, cells were serum-deprived for 2h, and then stimulated with IL-17A and lysed in buffer containing 1mM dimedone. Excess dimedone was removed using Micro Bio-spin 6 columns (Bio-Rad, Hercules, CA). Samples were analyzed by SDS-PAGE, and extent of sulfenic acid labeling was assessed using anti-dimedone antibody (Millipore, Darmstadt, Germany).

Nolin J.D., Tully J.E., Hoffman S.M., Guala A.S., van der Velden J.L., Poynter M.E., van der Vliet A., Anathy V, & Janssen-Heininger Y.M. (2014). The glutaredoxin/S-glutathionylation axis regulates interleukin 17A-induced pro-inflammatory responses in lung epithelial cells in association with S-glutathionylation of Nuclear Factor kappa B family proteins. Free radical biology & medicine, 0, 143-153.

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Profiling Protein Sulfenylation and Perthiosulfenylation

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For analysis of protein sulfenylation (Cys-SOH) and perthiosulfenylation (Cys-SSOH), cells were lysed in Western solubilization buffer (WSB) containing 1 mM DCP-bio1 (Kerafast or EMD Millipore Sigma), 200 U/mL catalase (Worthington, Lakewood, NJ) and 10 mM N-ethylmaleimide (Sigma) and incubated for 1 h on ice. Equal amounts of cell lysates were mixed with Laemli sample buffer, either in the presence or absence of 25 mmol/L DTT for 30 min, and separated by 10% SDS-PAGE for Western blotting with streptavidin-peroxidase (see below). For analysis of specific proteins of interest, excess DCP-bio1 reagent was removed from DCP-bio1-derivatized lysates by 6 successive washes with 20 mM Tris-HCl (pH 7.4) on Amicon Ultra-0.5 Centrifugal Filter Devices (Millipore). DCP-bio1-tagged proteins were subsequently collected with high capacity NeutrAvidin-agarose beads (Pierce) and washed successively with 1% SDS, 4 mol/L urea, and 1 mol/L NaCl [28] (link). Beads were then washed with 100 mmol/L ammonium bicarbonate either in the presence or absence of 10 mmol/L DTT for 30 min to assess the difference of sulfenic acid and perthiosulfenic acid tagged proteins.

Heppner D.E., Hristova M., Ida T., Mijuskovic A., Dustin C.M., Bogdándi V., Fukuto J.M., Dick T.P., Nagy P., Li J., Akaike T, & van der Vliet A. (2017). Cysteine perthiosulfenic acid (Cys-SSOH): A novel intermediate in thiol-based redox signaling?. Redox Biology, 14, 379-385.

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Redox Signaling Pathway Antibody Analysis

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Antibodies to phospho-p65 (ser536) (3033), total p65 (8242), Trx1
(2429), Trx2 (14907), HO-1 (5061), NQO1 (62262), TXNIP (c14715), SIRT6 (12486),
TBP (8515), Histone 3 (9715) and β-tubulin (2146) were purchased from
Cell Signaling Technology. Antibodies purchased from Abcam were to Prx1
(ab109506), Prx2 (ab109367, Prx3 (ab73349), Prx4 (ab59542), Prx6 (ab73350),
PrxSO_2/3 (ab16830), Nrf2 (ab62352), and β-actin (ab8226).
The antibody to LDH was purchased from Fitzgerald (20-LR22) and the antibody to
H3K9ac was from Millipore (06-942). The antibody to Srx was a kind gift from Dr.
Sue Gho Rhee (Yonsei University, Seoul, South Korea). The TXNIP antibody used
for IHC was from Proteintech (18243-1-AP). The antibody to AhpC was purified
from rabbit serum (33 (link)). Salmonella
typhimurium AhpC C165S protein was purified and expressed as previously
described (Poole et al, 1996) and AhpC C165S was reduced and labeled with biotin
maleimide as described (33 (link)). DCP-Bio1 was
purchased from Kerafast. Menadione, H₂O₂, EX-527,
N-ethylmaleimide (NEM), D-alanine, Flavin adenine dinucleotide disodium salt
hydrate (FAD), iodoacetamide (IAM), and catalase were purchased from Sigma
Aldrich. Dithiothreitol (DTT) was purchased from Life Technologies/Thermo Fisher
Scientific. Endotoxin-free recombinant fibronectin fragment (FN-f) was expressed
and purified as described (29 (link)).

Collins J.A., Kapustina M., Bolduc J.A., Pike J.F., Diekman B.O., Mix K., Chubinskaya S., Eroglu E., Michel T., Poole L.B., Furdui C.M, & Loeser R.F. (2021). Sirtuin 6 (SIRT6) regulates redox homeostasis and signaling events in human articular chondrocytes. Free radical biology & medicine, 166, 90-103.

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Adenanthin Stimulation of SMMC-7721 and HL-7702 Cells

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SMMC-7721 and HL-7702 cells were stimulated with 9 μM adenanthin at 37 °C for 1 h. Cells (1 × 10⁶) were collected and quenched with 500 μl lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% NP40, 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM IAA, 20 mM β-mercaptoethanol, 1 mM Na3VO4, 10 mM NEM, 100 μM DTPA and 200 U/ml catalase). The cell lysate was incubated on ice for 1 h, and then sonicated and centrifuged. DCP-Bio 1 (100 μM; EE0021, KeraFAST, Boston, MA, USA) were added into supernatant from the cell lysates. After 1 h incubation, the samples were collected for western blot.

Hou J.K., Huang Y., He W., Yan Z.W., Fan L., Liu M.H., Xiao W.L., Sun H.D, & Chen G.Q. (2014). Adenanthin targets peroxiredoxin I/II to kill hepatocellular carcinoma cells. Cell Death & Disease, 5(9), e1400-.

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Protein Sulfenylation Analysis in NCI-H292 Cells

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NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, were originally obtained from the American Type Culture Collection (ATCC), grown in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C and 5% CO₂, seeded at 100,000 cells/well in 24-well plates (Corning), and serum starved overnight.^{22 (link),25 (link)} For in situ analysis of protein sulfenylation (−SOH), cells were preloaded with 5 mM DYn-2 reagent^{21 (link)} (Kerafast) for 15–30 min, and subsequently stimulated with ATP (Sigma, St. Louis, MO; 100 µM) for 10 min. Cells were then lysed in chelator-free HEPES lysis buffer and clicked to biotin-azide (Kerafast) using established protocols (Supplementary Information). As an alternative approach to detect sulfenylated proteins, cells were lysed with western solubilization buffer containing 1 mM DCP-bio1 (Kerafast), as described previously^{24 (link),25 (link)}. FLAG-tagged Src and mutants were purified using Anti-FLAG M2 magnetic beads (Sigma) and eluted with 3× FLAG peptide (Sigma) as per manufacture’s protocol (Supplementary Information).

Heppner D.E., Dustin C.M., Liao C., Hristova M., Veith C., Little A.C., Ahlers B.A., White S.L., Deng B., Lam Y.W., Li J, & van der Vliet A. (2018). Direct cysteine sulfenylation drives activation of the Src kinase. Nature Communications, 9, 4522.

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YFP-OGG1 Localization and Oxidative Stress

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Cells expressing YFP or YFP‐OGG1were pretreated with or without 10 mM of NAC for 60 minutes then exposed to TNFα for 60 minutes. Whole cell lysates were made in an ice‐cold de‐oxygenized buffer containing 50 mM of Tris‐HCl (pH 7.5), 50 mM of NaCl, 1 mM of EDTA, 1 mM of EGTA, 1% of Nonidet P‐40, 2.5 mM of sodium pyrophosphate, protease inhibitor mixture (Sigma), 100 µM of diethylenetriaminepentaacetic acid, 5 mM of iodoacetamide, 200 units/mL catalase, and 0.1 mM of 3‐(2,4‐dioxocyclohexyl)propyl 5‐((3aR,6S,6aS)‐hexahydro‐2‐oxo‐1H‐thieno[3,4‐d]imidazol‐6‐yl) pentanoate (DCP‐Bio1; KeraFAST, Inc, Boston) as documented previously.²⁵, ³³ The supernatants of whole cell extracts were incubated with 30 µL of GFP beads at 4°C for 3 hours. The immunoprecipitates were resolved by SDS‐PAGE, and OGG1‐DCP‐Bio1 was detected by biotin antibody.

Hao W., Wang J., Zhang Y., Wang C., Xia L., Zhang W., Zafar M., Kang J., Wang R., Ali Bohio A., Pan L., Zeng X., Wei M., Boldogh I, & Ba X. (2020). Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription. The FASEB Journal, 34(6), 7427-7441.

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Sulfenic Acid Formation Analysis

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Sulfenic acid formation was examined using aLA samples (100 μM, in 10 mM phosphate buffer, pH 7.4) with or without photo-oxidation and glutathionylation treatment, and papain (100 μM, in 10 mM phosphate buffer, pH 7.4 treated with 10 mM H₂O₂) as a positive control. Protein samples were mixed with the sulfenic acid-selective chemical probe DCP-Bio1 (Kerafast, Boston, MA; 100-fold molar excess over protein concentration), then subjected to separation by SDS-PAGE and immunoblotting with a streptavidin–HRP conjugate.

Jiang S., Carroll L., Rasmussen L.M, & Davies M.J. (2020). Oxidation of protein disulfide bonds by singlet oxygen gives rise to glutathionylated proteins. Redox Biology, 38, 101822.

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EGFR Cysteine Redox Status Profiling

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Cysteine oxidation status of EGFR was determined by derivatization with sulfenic acid probe DCP-bio1 (Kerafast, Boston, MA), cell pre-loading with biotinylated glutathione ethyl ester (BioGEE), or derivatization with EZ-link Iodoacetyl-LC-biotin (Pierce, Rockford, Ill), to assess sulfenylation, S-glutathionylation, and cysteine thiol status, respectively. Biotin-tagged proteins were purified with NeutrAvidin-agarose beads and analyzed by Western blotting with α-EGFR.

Little A.C., Hristova M., van Lith L., Schiffers C., Dustin C.M., Habibovic A., Danyal K., Heppner D.E., Lin M.C., van der Velden J., Janssen-Heininger Y.M, & van der Vliet A. (2019). Dysregulated Redox Regulation Contributes to Nuclear EGFR Localization and Pathogenicity in Lung Cancer. Scientific Reports, 9, 4844.

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Sulfenylation Profiling of Cellular Proteins

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Sulfenylation was performed as described previously [22 (link)]. Briefly, cells were lysed in lysis buffer (50 mM HEPES, 50 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100) supplemented with 1 mM DCP-Bio1 (EE0028, Kerafast, Boston, MA, USA), 0.1 mM N-Ethylmalemide, 0.1 mM iodoacetamide and protease inhibitors. Samples were kept in ice for 30 min sonication and centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was transferred to a tube and rotated for 1 h at room temperature to allow for labeling of sulfonic acids. After incubation, protein was precipitated by acetone and centrifuged at 12,000 g for 5 min. The pellet was washed by 70% acetone and suspended in a non-supplemented lysis buffer. 1 mg of total protein was added to 50 μL slurry of streptavidin beads. Then, beads were rotated overnight at 4 °C. After 24 h, beads were centrifuged at 1000 g for 3 min, supernatant was discarded and beads were washed with 1 mL of lysis buffer. This washing step was repeated three times and beads were eluted in 30 μL of reducing 2 × LDS buffer (Life Technologies). For detection of sulfonation, anti-sulfonate antibody (ab176487, Abcam, Cambridge, MA, USA) was applied.

Lee H.Y., Kim H.K., Hoang T.H., Yang S., Kim H.R, & Chae H.J. (2020). The correlation of IRE1α oxidation with Nox4 activation in aging-associated vascular dysfunction. Redox Biology, 37, 101727.

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