Cell samples or whole zebrafish larvae (10 per sample) were snap-frozen using dry ice. Samples were analyzed via Western blot assay, as previously described [21 (link)], using anti-REP1 (2F1 clone, Millipore #MABN52, RRID:AB_10808665, Burlington, MA, USA) primary antibody diluted 1:1000 followed by secondary anti-mouse IgG HRP conjugate diluted 1:10,000 (Sigma, St. Louis, MO, USA) in blocking solution (5% dry milk, PBS/0.1% Tween [PBS-T]). The membrane was stripped and re-probed with 1:5000 anti-β-actin antibody (Sigma-Aldrich #A2228, RRID:AB_476697, St. Louis, MO, USA) or anti-vinculin (Santa Cruz Biotechnology #sc-25336, RRID:AB_628438) as a loading control. Three independent experiments were performed to determine the mean protein expression in fibroblast samples.
Anti mouse igg hrp conjugate
Manufactured by Merck Group
Sourced in United States
The Anti-mouse IgG HRP conjugate is a laboratory reagent used in various immunoassay techniques. It is composed of horseradish peroxidase (HRP) enzyme conjugated to anti-mouse immunoglobulin G (IgG) antibodies. This product can be used to detect and quantify mouse IgG in samples during research or diagnostic procedures.
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Lab products found in correlation
Anti 6xhis antibody, by Abcam (3 mentions)
Anti phospho p44 42 mapk antibody, by Cell Signaling Technology (2 mentions)
Anti mouse igg hrp conjugate, by Cell Signaling Technology (2 mentions)
Ecl prime western blot detection kit, by GE Healthcare (2 mentions)
Mouse monoclonal anti ha antibody, by Merck Group (2 mentions)
Mouse monoclonal gfp antibody, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bradford reagent, by Bio-Rad (2 mentions)
Las 1000, by Fujifilm (2 mentions)
A2228, by Merck Group (1 mentions)
Sc 25336, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp conjugate, by Merck Group (1 mentions)
Alexafluor 568 conjugate, by Thermo Fisher Scientific (1 mentions)
Powerwave 340, by Agilent Technologies (1 mentions)
9 protocols using anti mouse igg hrp conjugate
1
Western Blot Analysis of REP1 Protein
2
Sandwich ELISA for scFv Detection
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Sandwich ELISA was performed following standard method, using anti-6xHis antibody (Abcam, Cambridge, UK) 1:250 in PBS + 3%BSA to reveal the scFv, followed by anti-mouse IgG-HRP conjugate (Sigma, Missouri, USA) 1:500 in PBS + 3%BSA, as described in [35 (link)]. In case the ELISA assay is performed using mAb-hERG1, the procedure differs as the 2 h incubation with the monoclonal antibody is followed by the revealing using secondary peroxidate anti-mouse antibody (no intermediate incubation with anti-tag antibodies, e.g. anti-His, occurs).
3
Western Blot Analysis of Cellular Proteins
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Total cellular protein extractions were carried out as described previously 74 (link), and quantified using Bradford reagent (Bio-Rad), according to manufacturer’s instructions. Fifty μg of protein from each sample were resolved in a 12% (w/v) SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore). GFP-tagged strains were detected using 1:5,000 dilution of the mouse monoclonal GFP antibody (Santa Cruz Biotechnology) and secondary antibody anti-mouse IgG HRP conjugate (Cell Signaling Technology), at 1:10,000 dilution. For the HA-tagged proteins detection, a mouse monoclonal anti-HA antibody (Sigma) was used at 1:5,000 dilution as a primary antibody, followed by the same anti-mouse IgG HRP conjugate as a secondary antibody. The phosphorylated fractions of the MAP kinase, MpkA, were examined using anti-phospho p44/42 MAPK antibody (Cell Signaling Technologies) following the manufacturer’s instructions using a 1:2,000 dilution. The primary antibody was detected using an HRP-conjugated secondary antibody raised in rabbit (Sigma). Chemoluminescent detection was achieved using an ECL Prime Western Blot detection kit (GE HealthCare). To detect these signals on blotted membranes, the ECL Prime Western Blotting Detection System (GE Helthcare, Little Chalfont, UK) and LAS1000 (FUJIFILM, Tokyo, Japan) were used.
4
Pharmacokinetics of scFv-hERG1-Cys Antibody
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2 Balb/c mice have been injected with 160 μg of scFv-hERG1-Cys antibody and blood samples have been collected from the tail vein at 0, 5, 15, 30, 120, 360, 1440, 2880 minutes after antibody injection. Each sample was spun at 12000 rpm for 5 minutes; the resulting plasma was stored at −80° C until analyzed. The plasma concentration of scFv-hERG1-Cys antibody was determined by sandwich ELISA, using anti-6xHis antibody (Abcam, Cambridge, UK) 1:250 in PBS + 3%BSA to reveal the scFv, followed by anti-mouse IgG-HRP conjugate (Sigma, Missouri, USA) 1:500 in PBS + 3%BSA. The half-lives for elimination phase were determined using Origin 7.0 Software by fitting the last four data points into the first-order equation, T1/2 = (∆t/t1-t0)/∆C where (∆t/t1-t0) represents the slope of the curve and ∆C represents the value corresponding to the half of the antibody concentration at t1.
5
Western Blot Analysis of Protein Extracts
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Total cellular protein extractions were carried out as described71 (link), and quantified using Bradford reagent (Bio-Rad), according to manufacturer’s instructions. Fifty µg of protein from each sample were resolved in a 12% (w/v) SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore). GFP-tagged strains were detected using 1:5,000 dilution of the mouse monoclonal GFP antibody (Santa Cruz Biotechnology) and secondary antibody anti-mouse IgG HRP conjugate (Cell Signaling Technology), at 1:5,000 dilution. For the HA-tagged proteins detection, a mouse monoclonal anti-HA antibody (Sigma) was used at 1:5,000 dilution as a primary antibody, followed by the same anti-mouse IgG HRP conjugate as a secondary antibody. The phosphorylated fractions of the MAP kinase, MpkA, were examined using anti-phospho p44/42 MAPK antibody (Cell Signaling Technologies) following the manufacturer’s instructions using a 1:10,000 dilution. The primary antibody was detected using an HRP-conjugated secondary antibody raised in rabbit (Sigma). Chemoluminescent detection was achieved using an ECL Prime Western Blot detection kit (GE HealthCare). To detect these signals on blotted membranes, the ECL Prime Western Blotting Detection System (GE Helthcare, Little Chalfont, UK) and LAS1000 (FUJIFILM, Tokyo, Japan) were used.
6
ELISA Optimization for Monoclonal Antibody
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ELISA plates Immulon 2HB (Dynatech, USA) was coated with 100 μL/well of 0.0 to 40 μg/mL MoAb in borate buffer and incubated overnight at 4°C. The plates were then washed three times and non-specific binding sites were blocked with 1% skimmed milk in PBS-Tween 20 (0.05%) for 30 min at 37°C and then rinsed with 0.9%NaCl −0.05%Tween 20. An anti-mouse IgG-HRP conjugate (Sigma, A4416) was added at a 1:8,000 dilution in PBS-Tween 20, incubated for 2 h at 37°C and washed three times with 0.9%NaCl −0.05%Tween 20. The reaction was revealed with chromogen/substrate solution (0.05 M citrate/citric acid, 0.04 mg O-phenylenediamine and 0.12% H2O2). The reaction was stopped with H2SO4 and the absorbance was measured at 490 nm on an automatic ELISA reader Teca (Sunrise, Switzerland). The MoAb optimal concentration for the ELISA was 10.0 μg/mL (Figure 1 ).![]()
Determination of the optimal 1E4G4C2 MoAb concentration for the ELISA. 96-well polystyrene plate was coated with 0.0, 1.0, 2.5, 5.0, 10, 20 and 40 μg/mL of MoAb; non-specific binding sites were blocked with 1% skimmed milk; anti-mouse IgG-HRP conjugate was added at a 1:8,000 dilution and the reaction was revealed with chromogen/substrate solution. The results are shown as the arithmetic media ± standard deviation from three triplicate assays.
7
Multicolor Immunofluorescence and Western Blot
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Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate - A11008,
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 568 conjugate - A11031 all by Invitrogen;
Anti-Rabbit IgG, HRP conjugate - A0545,
Anti-Mouse IgG, HRP conjugate - A9044 all by Sigma-Aldrich.
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 568 conjugate - A11031 all by Invitrogen;
Anti-Rabbit IgG, HRP conjugate - A0545,
Anti-Mouse IgG, HRP conjugate - A9044 all by Sigma-Aldrich.
8
ELISA for Detecting Hybridoma Antibodies
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ELISA plate was coated with 100 µl of purified m-Ig (5 ng µl -1 ) in coating buffer. The plate was incubated overnight at 4 °C, washed thrice with PBST and blocked with 3% BSA in PBS at RT for 1 h. Thereafter, 100 µl of the hybridoma culture supernatant was added and incubated for 90 min at RT. Supernatant from SP2/0 myeloma cells served as negative control. Mouse serum from immunised mice at a dilution of 1:1000 was used as positive control. Mouse serum from immunised mice at a dilution of 1:1000 was used as positive control. The wells were washed with PBST followed by incubation with anti-mouse IgG HRP conjugate (Sigma, 1:2000 dilution in PBST) for 1 h at RT. Subsequently, 100 µl of the substrate solution containing ABTS (2, 2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) in sodium citrate buffer having 0.1% H 2 O 2 was added to the wells. After incubation for 10 min in dark, plate was read at 405 nm in ELISA plate reader (BioTek Power Wave 340, USA).
9
Pharmacokinetics of scFv-hERG1-Cys Antibody
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Athymic nude-Foxn1nu (nu/nu) (Envigo) mice were injected with 160 mg (8 mg/kg) of scFv-hERG1-Cys antibody and blood samples were collected from the tail vein at 0, 5, 15, 30, 120, 360, 1,440, and 2,880 minutes after antibody injection. Each sample was spun at 12,000 rpm for 5 minutes and the resulting plasma was stored at À80 C until analyzed. The plasma concentration of scFv-hERG1-Cys antibody was determined by sandwich ELISA (see Materials and Methods in the main text), using anti -6xHis antibody (Abcam) 1:250 in PBS þ 3% BSA to reveal the scDb-hERG1-b1, followed by anti-mouse IgG-HRP conjugate (Merck Sigma) 1:500 in PBS þ 3% BSA, as described in ref. 24 .The half lives for elimination phase were determined using Origin 7.0 Software by fitting the last four data points into the first-order equation, T1/2 ¼ (Dt/t1-t0)/DC where (Dt/t1-t0) represents the slope of the curve and DC represents the value corresponding to the half of the antibody concentration at t1, which corresponds to T ¼ 0. The serum stability against proteolytic activities of the scDb-hERG1-b1 was assessed using 20 mg of antibody. The scDb was incubated at 37 C in mouse serum up to 96 hours (0, 6, 24, 48, 72, 96 hours) and its concentration was determined through a sandwich ELISA assay.
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