To identify binding sites and cross-reactivity of the factor H (FH)-specific mAbs, human serum albumin was used as a negative control protein, and human FH, FH CCP1-4, FH CCP15 to 20, FHR-1, FHR-4B, and FHR-5 were immobilized at 4 μg/ml on Nunc MaxiSorp plates (Thermo Fisher Scientific). Monoclonal anti-FH antibodies were added at 1 μg/ml and their binding was detected with HRP-conjugated goat-anti-mouse IgG antibody (Dako) by the addition of TMB PLUS substrate (Kem-En-Tec Diagnostics). Absorbance was measured at 450 nm using a Thermo Multiskan EX microplate reader (Thermo Fisher Scientific).
Hrp conjugated goat anti mouse igg antibody
Manufactured by Agilent Technologies
The HRP-conjugated goat-anti-mouse IgG antibody is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is conjugated with horseradish peroxidase (HRP). This antibody can be used in various immunochemical techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect the presence and localization of mouse IgG proteins.
Automatically generated - may contain errors
Lab products found in correlation
Thermo multiskan ex microplate reader, by Thermo Fisher Scientific (1 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Ab8226, by Abcam (1 mentions)
Ecl reagent, by Promega (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Agilent Technologies (1 mentions)
Cellytic m cell lysis reagent, by Merck Group (1 mentions)
Amersham hyperfilm, by GE Healthcare (1 mentions)
Supersignal west pico chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Sartorius (1 mentions)
3 protocols using hrp conjugated goat anti mouse igg antibody
1
Characterizing FH-specific mAb Binding
2
Western Blot Analysis of CADM1 in GIST
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GIST-T1 cells, GIST-T1-CAD cells, a representative gastric GIST tissue, and a representative small intestinal GIST tissue were lysed in CelLytic M Cell Lysis Reagent (Sigma-Aldrich; Merck KGaA) containing 5 mM NAF, 1 mM Na3VO4, and proteinase inhibitor cocktail (Roche). As described previously (10 (link)), almost all gastric GISTs express a very low level of CADM1 protein and almost all small intestinal GISTs apparently express CADM1 protein. Western blot analysis was performed as previously reported (10 (link)). Briefly, anti-CADM1 chicken monoclonal antibody (clone. 3E1, MBL International), anti-KIT rabbit polyclonal antibody (A4502, Dako) or anti-β-actin mouse monoclonal antibody (ab8226, Abcam) were used for primary antibodies after electrophoresis and membrane transfer. Then, membranes were incubated either with horse radish peroxidase (HRP)-conjugated donkey anti-chicken IgY antibody (EMD Millipore, Sigma-Aldrich; Merck KGaA), HRP-conjugated goat anti-rabbit IgG antibody, or HRP-conjugated goat anti-mouse IgG antibody (Dako) after the electrophoresis and membrane transfer. Proteins of interest were then visualized by incubation with enhanced chemiluminescence (ECL) reagent (Promega).
3
TARDBP Protein Expression Quantification
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Proteins electrophoresed through a 10% SDS-polyacrylamide gel were transferred onto a nitrocellulose membrane (Sartorius) at 4 °C overnight (20 V) using a Western Transfer Unit (BioRad). The membrane was then blocked for 1 h at room temperature (RT) with blocking buffer (5% (w/v) skim milk in PBS containing 0.1% (v/v) TX-100). The membrane was then incubated with TARDBP monoclonal antibody (clone 2E2-D3, Abnova; 1:500) followed by an HRP-conjugated goat-anti-mouse IgG antibody (DAKO; 1:2000). Each antibody was diluted into blocking buffer, and each incubation was followed by washing the membrane 3X with PBS containing 0.1% (v/v) TX-100, followed by 3X washes with PBS. Bound antibodies were detected using Supersignal West Pico Chemiluminescence Substrate (Thermo Fisher Scientific), according to the manufacturer’s instructions. Bands were detected using X-ray film (Amersham Hyperfilm; GE Life Sciences).
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