Ab71940

Manufactured by Abcam

Sourced in United States

Ab71940 is a protein that binds to a specific target. It can be used for various laboratory applications.

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Lab products found in correlation

6 protocols using ab71940

Quantifying C1q Binding to Pneumococci

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To investigate the binding of C1q to pneumococci, bacteria were grown in THY broth to the early exponential phase (OD_600nm ~ 0.3), washed in TBS, and distributed in 96‐well plates (5 × 10⁷ CFU/well). Afterward, wells were blocked with TBS‐2% milk and incubated with the indicated doses of human recombinant C1q (Calbiochem; 204,876) for 1 h at 37°C. Cell‐surface bound C1q was revealed with a mouse anti‐C1q monoclonal antibody (Abcam; ab71940) diluted 1:100 in TBS‐1% milk followed by incubation with AP‐conjugated anti‐mouse IgG (Sigma‐Aldrich; A7434).
For flow cytometry immunofluorescence analysis, bacteria were incubated with recombinant human C1q (5 μg/ml) for 1 h at room temperature in constant agitation. Washed bacteria were incubated with anti‐C1q antibody (Abcam; ab71940) diluted 1:100 in 1% milk‐PBS for 1 h at room temperature in constant agitation. Afterward, to detect the binding of C1q to the bacteria, an Alexa‐Fluor‐488‐labeled anti‐mouse antibody was used. Bacteria were analyzed with a Flow Cytometer (BD FACS canto II) using the Flowlogic software.

De Gaetano G.V., Coppolino F., Lentini G., Famà A., Cullotta C., Raffaele I., Motta C., Teti G., Speziale P., Pietrocola G, & Beninati C. (2022). Streptococcus pneumoniae binds collagens and C1q via the SSURE repeats of the PfbB adhesin. Molecular Microbiology, 117(6), 1479-1492.

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Quantitative Protein Analysis from Serum

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GSK3050002 isolated from serum by anti-idiotype affinity column were further fractionated and purified using a Superdex 200 10/300 column on an FPLC. For Western blot analysis, approximately 1 μg of protein was reduced (NuPAGE reducing agent) and denatured (Novex SDS sample buffer). C1q, C3 and IgG were detected using antibodies (Abcam; ab71940, Abcam; ab196639 and JIR 315-165-045, respectively) at 1 μg/ml and standard curves from 1 to 5000 ng for each recombinant protein run on the same SDS PAGE gel.

Laffan S.B., Thomson A.S., Mai S., Fishman C., Kambara T., Nistala K., Raymond J.T., Chen S., Ramani T., Pageon L., Polsky R., Watkins M., Ottolangui G., White J.R., Maier C., Herdman M, & Bouma G. (2020). Immune complex disease in a chronic monkey study with a humanised, therapeutic antibody against CCL20 is associated with complement-containing drug aggregates. PLoS ONE, 15(4), e0231655.

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Comprehensive Protein Expression Analysis

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At first, the cell or tissue protein were extracted using the RIPA lysis buffer (P0013B, Beyotime). The protein concentration was determined by the BCA protein assay kit. All samples were prepared at a final concentration of 1 μg/μl in loading buffer (CW0027S, CwBio). Protein samples (20 μg) were loaded into a 10–15% SDS-polyacrylamide gel in the Bio-Rad Electrophoresis System to separate the proteins with different molecular weight. Then, the proteins in the gel were transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% bovine serum albumin (BSA) solution, the membranes were incubated with primary antibodies (Integrin-α_L, ab186873, Abcam; Integrin-β₂, ab131044, Abcam; C1q, ab71940, Abcam; Integrin-β₃, ab75872, Abcam; CD11b, ab133357, Abcam; CD45, ab10558, Abcam; cleaved caspase-3, #9661s, Cell signaling technology; CD44, ab189524, Abcam; HO-1, ab68477, Abcam; β-tubulin, CW0098, CwBio; iNOS, ab15323, Abcam; CD206, ab125028, Abcam; Arginase-1, 93668s, Cell signaling technology; Histone H3, #9715, Cell signaling technology; GM130, 610,822, BD Biosciences) at 4 °C overnight. After incubation with the corresponding secondary antibodies, the PVDF membranes were imaged using Western chemiluminescent horseradish peroxidase (HRP) substrate (Millipore) with an imaging system (Tanon 4600, Shanghai).

Bao L., Dou G., Tian R., Lv Y., Ding F., Liu S., Zhao R., Zhao L., Zhou J., Weng L., Dong Y., Li B., Liu S., Chen X, & Jin Y. (2021). Engineered neutrophil apoptotic bodies ameliorate myocardial infarction by promoting macrophage efferocytosis and inflammation resolution. Bioactive Materials, 9, 183-197.

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Western Blot Analysis of Raphe Nuclei Proteins

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The protein expression levels of Iba-1, C1q, cleaved caspase 3, synaptophysin, PSD95, and β-actin in raphe nuclei homogenates were evaluated using Western blotting. The protein concentrations of the homogenates were equalized, and the samples were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. To minimize nonspecific binding, the membranes were blocked in 5% bovine serum albumin for 1 hour. The membranes were incubated overnight at 4°C with the following primary antibodies: Iba-1 (1:500; 061-20001, Wako Biologicals), C1q (1:50; ab71940, Abcam), cleaved caspase 3 (1:200; no. 9664S, Cell Signaling Technology), PSD95 (1:200; ab13552, Abcam), synaptophysin (1:500; ab8049, Abcam), or β-actin (1:2500; PA1-183, Thermo Fisher Scientific). After washing, the membranes were incubated with an HRP-conjugated anti-rabbit or anti-mouse antibody (GeneTex Inc., Irvine, CA) for 1 hour. The Western blotting results were visualized with an enhanced chemiluminescence advanced kit. The intensity was analyzed by ImageJ version 1.46 (NIH, Bethesda, MD, USA).

Lee J.S., Lee S.B., Kim D.W., Shin N., Jeong S.J., Yang C.H, & Son C.G. (2021). Social isolation–related depression accelerates ethanol intake via microglia-derived neuroinflammation. Science Advances, 7(45), eabj3400.

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Immunofluorescence Staining of Brain Slices

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Immunofluorescence staining was conducted as described previously (19 (link)). Coronal sections were washed with PBS and incubated with 5% BSA and 0.3% Triton X-100 for 1 h at room temperature. The brain slices were then incubated with primary antibodies overnight at 4 °C. The primary antibodies in our study were as follows: goat anti-Iba-1 antibody (1:500, ab5076, Abcam), mouse anti-NeuN antibody (1:500, ab104224, Abcam), mouse anti-GFAP antibody (1:500, ab10062, Abcam), rat anti-CR3/CD11b antibody antibody (1:300, ab8878, Abcam), mouse anti-C1q antibody (1:300, ab71940, Abcam), and rabbit anti-CD163 antibody (1:200, ab182422, Abcam). The brain slices were incubated with secondary antibodies at room temperature for 2 h. Finally, three random fields of vision were examined in each slice to acquire the average number of positive cells using a fluorescence microscope (Leica, Mannheim, Germany).

Zheng Y., Fan L., Xia S., Yang Q., Zhang Z., Chen H., Zeng H., Fu X., Peng Y., Xu C., Yu K., Liu F, & Cao S. (2022). Role of complement C1q/C3-CR3 signaling in brain injury after experimental intracerebral hemorrhage and the effect of minocycline treatment. Frontiers in Immunology, 13, 919444.

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Immunolocalization of C1q using Monoclonal Antibody

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An extensively characterized mouse monoclonal C1q (JL-1) antibody reactive against the collagen-like region (CLR) obtained commercially (ab71940; Abcam, Cambridge, MA, USA) was used for immunoEM studies at 1:200 dilution. The antibody was generated by immunization of C1q^−/− C57BL/6 mice with purified mouse C1q. The antibody has been previously shown to recognize C1q with a high degree of specificity in a myriad of different tissues and cell types by immunoblotting, immunohistochemistry, and immunofluorescence procedures [27 (link)–30 (link)]. The C1q antibody detects a band migrating at ~ 26 kDa in immunoblots. Normal sera and IgG-free BSA were purchased from Jackson Immunoresearch (West Grove, PA, USA). All chemicals and supplies for electron microscopy were purchased from Sigma Aldrich (St. Louis, MO, USA) and Electron Microscopy Sciences (Hatfield, PA, USA), respectively.

Datta D., Leslie S.N., Morozov Y.M., Duque A., Rakic P., van Dyck C.H., Nairn A.C, & Arnsten A.F. (2020). Classical complement cascade initiating C1q protein within neurons in the aged rhesus macaque dorsolateral prefrontal cortex. Journal of Neuroinflammation, 17, 8.

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