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Peroxidase labeling kit nh2

Manufactured by Dojindo Laboratories
Sourced in Japan

The Peroxidase Labeling Kit-NH2 from Dojindo Laboratories is a product designed for the labeling of amino-modified biomolecules, such as proteins, with peroxidase. The kit contains the necessary reagents and instructions to facilitate the covalent attachment of peroxidase to the target biomolecule, enabling its use in various immunoassay and detection applications.

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26 protocols using peroxidase labeling kit nh2

1

Myc-GAPDH Protein Detection

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Chemical compounds and crystallization reagents were purchased from Nacalai Tesque, Inc. and Hampton Research Corp, respectively. Anti-myc (05-724) and anti-GAPDH (MAB374) antibodies were purchased from Millipore. DSA lectin (J105; J-Chemical) was labeled with HRP using a peroxidase labeling kit – NH2 (DOJINDO), following the manufacturer’s procedure.
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2

Monoclonal Antibody Purification and Labeling

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Three hybridoma cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum. The monoclonal antibodies were purified from the culture supernatants using an Ex-Pure Protein G kit (Kyoto Monotech, Kyoto, Japan). Purified antibodies were labeled with HRP using the Peroxidase Labeling Kit NH2 (Dojindo Laboratories, Kumamoto, Japan). The concentrations of the monoclonal antibodies were determined from the absorbance at 280 nm assuming an E2801% = 10.0.
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3

Quantitative Sandwich-ELISA for Stx2

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Sandwich-ELISA was used to detect Stx2 in the bacterial culture supernatants, and was carried out in RIDASCREEN Verotoxin microtiter plates (R-Biopharm GmbH, Darmstadt, Germany) coated with capture antibodies for detecting both Stx1 and Stx2. A monoclonal antibody against Stx2 (LSBio, WA, USA) conjugated with horseradish peroxidase using Peroxidase Labeling Kit–NH2 (Dojindo, Kumamoto, Japan) was used as the detection antibody. A standard curve was determined with 2-fold serial dilutions of purified Stx2 (provided by Denka Seiken Co. Ltd.). Detection was carried out with urea peroxidase/TMB substrate reagent and 1 M sulfuric acid as the stop reagent, both were provided in the RIDASCREEN Verotoxin kit. Absorbance at 450 nm (A450) was measured using a 96-well plate reader (Multiscan FC, Thermo Fisher Scientific, MA, USA).
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4

Antibodies and Lectins for Cell Analysis

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The following antibodies and lectins were used: anti-myc (mouse, clone 4A6; Millipore; catalog no.: 05-724), anti-BACE1 (mouse, clone 1A11, generous gift from Dr Bart De Strooper (40 (link))), anti-GAPDH (mouse, clone 6C5; Merck Millipore; catalog no.: MAB374), anti-Golgin-97 (rabbit, clone D8P2K, Cell Signaling Technology; catalog no.: 13192S), anticalnexin (rabbit, abcam; catalog no.: ab22595), anti-N-cadherin (rabbit, abcam; catalog no.: ab18203), horseradish peroxidase (HRP)–conjugated antimouse IgG (GE Healthcare; catalog no.: NA931V), HRP-conjugated anti-rabbit IgG (GE Healthcare, Amersham; catalog no.: NA934V), DSA (J-Chemical; catalog no.: J105), SSA letin (J-Chemical; catalog no.: J118), biotinylated RCA (Vector Laboratories; catalog no.: B-1085), Alexa546-conjugated anti-rabbit IgG (Invitrogen; catalog no.: A10040), and Alexa488-conjugated antimouse IgG (Invitrogen; catalog no.: A21202). DSA and SSA were conjugated to HRP using a Peroxidase Labeling Kit-NH2 (Dojindo), according to the manufacturer’s protocol.
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5

Quantification of Shiga Toxin 2

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Bacterial cells were inoculated into 40 ml of lysogeny broth and grown to mid-log phase at 37 °C with shaking. MMC was then added to the culture at a final concentration of 500 ng/ml. At 5 h after MMC addition, 100 µl of the culture was collected and immediately subjected to sonication using a Bioruptor (Cosmo Bio, Tokyo, Japan). The soluble fractions of each cell lysate were obtained via centrifugation at 14,000 × g at 4 °C for 10 min. The Stx2 concentration in each cell lysate was determined using a previously described sandwich ELISA [69 (link)] using RIDASCREEN Verotoxin microtiter plates (R-Biopharm GmbH, Germany) coated with capture antibodies that recognize Stx2 and monoclonal antibodies against Stx2 (LSBio, WA, USA) conjugated with horseradish peroxidase using a Peroxidase Labeling Kit–NH2 (Dojindo, Kumamoto, Japan) as detection antibodies. Note that both Stx2a and Stx2g could be quantified at a certain level of accuracy using the detection antibodies [70 (link)].
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6

AGE-BSA Binding to Collagen Assay

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AGE-BSA (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was conjugated with horseradish peroxidase (HRP) using a peroxidase labeling kit-NH2 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). HRP-labeled AGE-BSA was incubated with or without OSSC1E-K19 or AG in collagen-coated microtiter plates. HRP-labeled AGE-BSA was incubated to react with collagen at 37°C for 4 h. After washing with a solution of 0.05% Triton-X 100 in phosphate-buffered saline (PBS), 100 µl 3,3′,5,5′-tetramethylbenzidine chromogen (cat. no. T4444; Sigma-Aldrich) was added followed by incubation for 15 min. The absorbance at 450 nm was determined using an absorbance plate reader (Synergy HT) after the addition of 40 µl 1 N H2SO4.
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7

AGE-RAGE Interaction Assay Protocol

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AGE-BSA was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and labeled with horseradish peroxidase using a kit (Peroxidase Labeling Kit-NH2, Dojindo Molecular Technologies, Inc., Tokyo, Japan). The interaction between AGE and RAGE was assayed using a protocol reported earlier [30 (link),31 (link),32 (link)]. Briefly, AGE-BSA and the test compounds were added to RAGE-coated, 96-well plates. The binding of the AGE-BSA to RAGE was determined using a 3,3′,5,5′-tetramethylbenzidine enzyme-linked immunosorbent assays (TMB-ELISA) substrate solution (Sigma). The inhibition of the AGE/RAGE interaction is expressed as the percentage of the binding of the AGE-BSA with RAGE in the presence of the test compounds.
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8

Quantitative Detection of HPV16 E7 Oncoprotein

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A double-antibody sandwich ELISA assay was used for pairing experiments. Unlabeled mAbs were used as capture antibodies, HRP-labeled mAbs as detection antibodies, and TMB substrate was used for coloration. The OD450 nm value was used to indirectly measure the antigen content. HRP was used to label 8 high effective mouse anti-HPV16 E7-HIS oncoprotein mAbs according to the instructions of the Peroxidase Labeling Kit-NH2 (Dojindo, Sahnghai, China). HRP-labeled mAbs were used as detection antibodies, and mAb pairing experiments were performed with unlabeled mAbs as capture antibodies. A total of 56 pairs were paired. The pair of mAbs with the strongest pairing signal was subjected to conventional double-antibody sandwich ELISA to quantitatively detect the lowest limitation of the HPV16 E7-HIS oncoprotein.
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9

Fibrin Clot Immunoassay Protocol

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One microgram of fibrinogen (Merck, Darmstadt, Germany) was immobilized onto a 96-well plate for 12 h. The fibrinogen-immobilized plates were then treated with a thrombin solution at 37 °C for 1 h to prepare fibrin clot plates. The wells were then blocked using N102 (Nichiyu, Tokyo, Japan) for 2 days. insoluble fibrin mAb and control mAb were conjugated to horseradish peroxidase (HRP) using Peroxidase Labeling Kit–NH2 (Dojindo Molecular Technologies, Kumamoto, Japan) and diluted with PBS containing 1% Block Ace (KAC Hyougo, Japan) at 0.25–1 μg/ml. Subsequently, the antigen-coated plates were incubated in diluted HRP-labeled mAbs for 1 h and the wells were washed with Tris-buffered saline (TBS) containing 0.05% Tween 20. Finally, the mAbs bound to the wells were visualized using a 1-Step Slow TMB-ELISA (Thermo, Massachusetts, USA) as a substrate for 5 min.
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10

Antibody and Lectin Toolkit for Cell Biology

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The following antibodies and lectins were used: anti-GnT-V (mouse, clone 706824, R&D Systems), anti-α1 sodium potassium ATPase (mouse, clone 464.6, Abcam), anti-LAMP1 (rabbit, Abcam), anti-GAPDH (mouse, clone 6C5, Merck Millipore), anti-GRP78 (BiP) (rabbit, Abcam), anti-GM130 (rabbit, clone D6B1) (Cell Signaling Technology), anti-Golgin97 (rabbit, clone D8P2K, Cell Signaling Technology), anti-Rab5 (rabbit, clone C8B1, Cell Signaling Technology), anti-Rab7 (rabbit, Cell Signaling Technology), anti-Rab11 (rabbit, Cell Signaling Technology), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (GE Healthcare), HRP-conjugated anti-rabbit IgG (GE Healthcare), unconjugated-L4-PHA (J-Chemical), biotinylated-SSA (J-Chemical), biotinylated-RCA (Vector Laboratories), FITC-conjugated L4-PHA (J-Oil Mills), Rhodamine-conjugated L4-PHA (Vector Laboratories), Alexa546-conjugated anti-mouse IgG (Invitrogen), Alexa488-conjugated anti-rabbit IgG (Invitrogen). L4-PHA was conjugated to HRP using a Peroxidase Labeling Kit-NH2 (Dojindo) as described previously (22 (link)).
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