The antigens aHSA (≈60% isoAsp) and fHSA (~0% isoAsp) were diluted to 10 µg/mL by Phosphate Buffered Saline (PBS) (Cytiva, Marlborough, MA, USA), pH 7.4. The 96-well white opaque polystyrene plates were coated with 50 µL/well of the aHSA and fHSA diluted in PBS and incubated at RT for 2 h. Plates were washed in 300 µL/well PBS containing 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) (PBST) three times and shaken dry. The vacant sites were blocked with 200 µL/well 10% skim milk powder (Sigma-Aldrich, St. Louis, MO, USA) in PBST at 4 °C for overnight and washed with PBST as above. Then 50 µL/well of mAb as primary antibody in blocking buffer was added and incubated at RT for 2 h. After washing with PBST, a goat anti-mouse IgG Conjugated to Horseradish Peroxidase (Jackson Immuno Research, Cambridge, UK) was used as secondary antibody and incubated at RT for 2 h. Working solution (100 µL/well) was prepared according to the SuperSignal ELISA Pico Chemiluminescent Substrate protocol (Thermo Scientific, Waltham, MA, USA), and chemiluminescence intensities were measured immediately by the luminometer Tecan Infinite M200 Pro (Tecan, Männedorf, Switzerland).
Goat anti mouse igg conjugated to horseradish peroxidase
Manufactured by Jackson ImmunoResearch
Sourced in United Kingdom, Cameroon
Goat anti-mouse IgG conjugated to horseradish peroxidase is a secondary antibody reagent used for detection and quantification in immunoassays. The goat-derived antibody specifically recognizes and binds to mouse immunoglobulin G (IgG), and the horseradish peroxidase enzyme is used as a reporter for signal generation.
Automatically generated - may contain errors
Lab products found in correlation
Skim milk powder, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Phosphate buffered saline (pbs), by Cytiva (1 mentions)
Lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Mab1501, by Merck Group (1 mentions)
Image studio for c digit, by LI COR (1 mentions)
Mmp 13, by R&D Systems (1 mentions)
5 protocols using goat anti mouse igg conjugated to horseradish peroxidase
1
Quantification of Modified Protein Antigens by ELISA
2
Semiquantitative Immunoblotting of TJ Proteins
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TJ protein levels were tested using semiquantitative immunoblotting. HK-2 cells were plated onto 100 mm-Petri dishes at 0.5 ~ 1 × 105 cells/mL and cultured for siRNA transfection. Cells were harvested and lysed in lysis buffer (Santacruz, Heidelberg, Germany) containing protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min on ice. Cell lysates were centrifuged at 14,000 x g for 20 min at 4°C and protein concentrations measured using Bradford protein assay kits (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were electrophoresed on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes and blocked in 5% skim milk in PBST (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h. Membranes were probed overnight at 4°C with primary antibodies: rabbit polyclonal anti-occludin, rabbit polyclonal anti-ZO-1 or mouse monoclonal anti-claudin-2 (Zymed Labs, Jerusalem, Israel). Secondary antibodies were goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Sites of antibody-antigen reaction were viewed using enhanced chemiluminescence (GenDEPOT, Barker, TX), and band densities were quantified by densitometry using a laser scanner and Quantity One Software (Basic version 4.6.9, Bio-Rad, Hercules, CA).
3
Validating CyaA Fusion Proteins in L. pneumophila
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The formation of a CyaA fusion proteins in L. pneumophila was validated by Western blot analysis using the primary anti CyaA antibody 3D1 (Santa Cruz Biotechnology, Inc.) diluted 1:500 and goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, Inc.) diluted 1:10,000 as the secondary antibody.
4
Quantitative Protein Expression Analysis
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Whole cell protein lysates (25 μg) were electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose membranes and probed with primary antibodies against MMP-2 and RANKL (sc-58386 and -377079, respectively, Santa Cruz Biotechnology), MMP-9 (D603H, Cell Signaling), MMP-13 (MAB511, R&D systems), and actin (MAB1501, Millipore). Goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch) were used as secondary antibodies. Quantitative evaluation was performed by densitometry using the Scanner C-DiGit (LI-COR) and the software Image Studio for C-Digit. Intensities from each band were normalized to actin. Statistical analysis among transfected groups was made by U Mann Whitney test, considering a significance of p < 0.05.
5
Detecting Toxin in Biofilm Extracts
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To detect toxin in biofilm matrix extracts, we first measured the concentration of total protein in the matrix extract (using the BCA Protein Assay Kit, Pierce, Rockford, IL). For Western blot 5 µg of extract was fractionated on a 6% SDS-PA gel. After electrotransfer to a nitrocellulose membrane, we blocked the membrane overnight at 4°C in PBS with 5% nonfat milk and 0.1% Tween 20. The membrane was probed with a 1∶1000 dilution of mouse anti-toxin antibody obtained from C. difficile infected mice [43] . Bands were visualized by application of a secondary goat anti-mouse IgG conjugated to horse radish peroxidase (Jackson Immuno-Research, West Grove, PA) and the SuperSignal West Pico Chemiluminscent Substrate system (Pierce, Rockford, IL).
Total toxin (A and B) production was measured using the Wampole C.difficile TOX A/B II kit (Tech Labs Inc., Blacksburg, VA) according to the manufacturer instructions. EPS extracts containing 1 µg of total protein were diluted with the supplied diluent and analyzed in triplicates. Total toxin level was determined by measuring A450/OD620. We analyzed biofilms from three independent inoculations. Data was analyzed using one-way ANOVA and the two sample Student's T test (XLSTAT). P values <0.05 were considered statistically significant.
Total toxin (A and B) production was measured using the Wampole C.difficile TOX A/B II kit (Tech Labs Inc., Blacksburg, VA) according to the manufacturer instructions. EPS extracts containing 1 µg of total protein were diluted with the supplied diluent and analyzed in triplicates. Total toxin level was determined by measuring A450/OD620. We analyzed biofilms from three independent inoculations. Data was analyzed using one-way ANOVA and the two sample Student's T test (XLSTAT). P values <0.05 were considered statistically significant.
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