Competitive BPM labeling assay was performed as described previously43 (link). Briefly, mouse plasma (20 μg of protein) were prepared and mixed with biotin-PEAC5-maleimide (BPM, Dojindo) (25 µM) and rocked at 37 °C for 30 min to label cysteine free-thiols on proteins. Then the plasma was incubated with Laemmli buffer and boiled at 95 °C for 5 min. The proteins were subjected to SDS-PAGE, and BPM-bound proteins were visualized using anti-biotin-HRP-linked antibody (#7075, Cell signaling). For identification of Gpx3, the gel was stained with coomasie brilliant blue (Nacalai), and then dehydrated by 30% acetonitrile, and proteins were reduced by dithiothreitol (100 mM), alkylated by iodoacetamide (100 mM) and digested by Trypsin-Gold (Promega) with protease MAX surfactant (Promega). Peptides were analyzed with liquid chromatography–mass spectrometry (EASY-nLC1000 and Orbitrap Elite, Thermo). Data were analyzed by Mascot ver.2.5.1 (Matrix science).
Biotin peac5 maleimide bpm
Manufactured by Dojindo Laboratories
Sourced in Japan
Biotin-PEAC5-maleimide (BPM) is a bioconjugation reagent used for the covalent attachment of biotinylated molecules to other compounds containing a free sulfhydryl group. It provides a stable linker between the biotin moiety and the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Mascot ver2, by Matrix Science (1 mentions)
Anti biotin hrp linked antibody, by Cell Signaling Technology (1 mentions)
Trypsin gold, by Promega (1 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (1 mentions)
Proteasemax surfactant, by Promega (1 mentions)
Na2s4, by Dojindo Laboratories (1 mentions)
Anti gapdh antibody, by Fujifilm (1 mentions)
Escherichia coli bl21 cells, by Promega (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Anti rabbit antibodies, by Cell Signaling Technology (1 mentions)
Anti mouse igg secondary antibody, by Cell Signaling Technology (1 mentions)
Anti phospho egfr tyr1068 antibody, by Cell Signaling Technology (1 mentions)
Anti p44 42 map kinase antibody, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Anti egfr antibody, by Cell Signaling Technology (1 mentions)
Anti mouse igg 7076 secondary antibody, by Cell Signaling Technology (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Dojindo Laboratories (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
3 protocols using biotin peac5 maleimide bpm
1
Competitive BPM Labeling for Cysteine Proteomics
2
Investigating Protein Interactions Using Biotin-Maleimide
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Dimethyl sulfoxide (DMSO), 1,4-NQ (98% purity determined by gas chromatography) and anti-GAPDH antibody were from Wako Pure Chemical Industries (Osaka, Japan), Tokyo Chemical Industries (Tokyo, Japan) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Biotin-PEAC5-maleimide (BPM) and Na2S4 were from Dojindo Laboratories (Kumamoto, Japan). Dynabeads M-280-sheep anti-rabbit immunoglobulin G (IgG) was from Invitrogen (Carlsbad, CA, USA). Anti-Akt, anti-CREB, anti-phosphorylated Akt (Ser473), anti-phosphorylated CREB (Ser133), horseradish peroxidase (HRP)-conjugated anti-biotin antibodies, anti-rabbit antibodies and anti-mouse IgG secondary antibodies were from Cell Signaling Technology (Beverly, MA, USA). Escherichia coli BL21 cells and trypsin were from Promega Co. (Madison, WI, USA). Glutathione 4B Sepharose was from GE Healthcare (Chicago, IL, USA). All other reagents were of the highest purity available.
3
Quantification of Cellular Redox Signaling
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Epidermal growth factor (EGF), p-nitrophenyl phosphate (pNPP), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1,2-NQ (95% purity determined by HPLC), 1,4-NQ (100% purity determined by GC), and 1,4-BQ (99.1% purity determined by iodometric titration) were purchased from Tokyo Chemical Industry (Tokyo, Japan).
PD153035 and anti-PTP1B antibody (# PH01) were acquired from Calbiochem (San Diego, CA, USA). Biotin-(PEAC) 5 -maleimide (BPM) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Dojindo Laboratories (Kumamoto, Japan). The anti-EGFR antibody (#2232), anti-phospho EGFR Tyr1068 antibody (#2234), anti-p44/42 MAP kinase antibody [anti-extracellular signal-regulated kinase 1/2 (ERK1/2), #9102], anti-phospho ERK1/2 antibody (#9101), horseradish peroxidase (HRP)-linked anti-rabbit IgG (#7074), and anti-mouse IgG (#7076) secondary antibody were procured from Cell Signaling Technology (Beverly, MA, USA). Fetal bovine serum, GlutaMAX-I, tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and Ni-IDA ProBond were from Corning (Woodland, CA, USA), Gibco (Grand Island, NY, USA), Hampton Research (Aliso Viejo, CA, USA) and Invitrogen (Carlsbad, CA, USA), respectively. The anti-1,4-NQ antibody was prepared as previously reported (Hirose et al., 2012) . All other reagents used were of the highest purity available.
PD153035 and anti-PTP1B antibody (# PH01) were acquired from Calbiochem (San Diego, CA, USA). Biotin-(PEAC) 5 -maleimide (BPM) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Dojindo Laboratories (Kumamoto, Japan). The anti-EGFR antibody (#2232), anti-phospho EGFR Tyr1068 antibody (#2234), anti-p44/42 MAP kinase antibody [anti-extracellular signal-regulated kinase 1/2 (ERK1/2), #9102], anti-phospho ERK1/2 antibody (#9101), horseradish peroxidase (HRP)-linked anti-rabbit IgG (#7074), and anti-mouse IgG (#7076) secondary antibody were procured from Cell Signaling Technology (Beverly, MA, USA). Fetal bovine serum, GlutaMAX-I, tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and Ni-IDA ProBond were from Corning (Woodland, CA, USA), Gibco (Grand Island, NY, USA), Hampton Research (Aliso Viejo, CA, USA) and Invitrogen (Carlsbad, CA, USA), respectively. The anti-1,4-NQ antibody was prepared as previously reported (Hirose et al., 2012) . All other reagents used were of the highest purity available.
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