Human c1q

Manufactured by Merck Group

Sourced in United States

Human C1q is a laboratory product that is a component of the complement system, a group of proteins involved in the immune response. It plays a crucial role in the activation of the classical complement pathway. The product is intended for use in research and scientific investigations.

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9 protocols using human c1q

Quantifying Serum Antibodies and Immune Complexes

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Blood urea nitrogen concentrations were measured with a Beckman Autoanalyzer (Beckman Coulter, Fullerton, CA). Serum anti-apoferritin IgG antibodies were measured by ELISA.^{9 (link)} Briefly, 96-well plates were coated with apoferritin. Serial dilutions of sera were plated and incubated at room temperature for 2 hours, followed by HRP-conjugated goat anti-mouse IgG (Kierkegard & Perry Laboratories, Gaithersburg, MD) and OPD peroxidase substrate (Sigma-Aldrich, St. Louis, MO). The OD₄₅₀ was then measured.
Circulating IC levels were measured by C1q binding ELISA as described previously.^{41 (link)} Briefly, 96-well plates were coated with 10 μg/ml human C1q (Sigma) in carbonate buffer. After blocking with 1% BSA, sera samples were loaded in serial dilutions starting at 1/1000, followed by HRP-goat anti-mouse IgG (Sigma, 1:2000) and then TMB (Jackson ImmunoResearch Laboratories, West Grove, PA). The OD₄₅₀ was then measured. Sera samples from unmanipulated wildtype mice were used as negative control.

Alexander J.J., Chaves L.D., Chang A., Jacob A., Ritchie M, & Quigg R.J. (2015). CD11b is protective in complement-mediated immune complex glomerulonephritis. Kidney International, 87(5), 930-939.

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Investigating C1q and TGF-β effects on LX2 cells

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LX2 cells were grown and maintained in Dulbecco's modified Eagle's medium (Gibco, Camarillo, CA, USA) supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences), 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid (HEPES; Gibco), sodium pyruvate (Gibco), and penicillin and streptomycin (Gibco) at 37°C in a 5% CO₂ incubator. LX2 cells were cultured in medium without serum for 24 hours, then treated with 100 μg/ml of human C1q purified from human serum (Sigma‐Aldrich, Saint Louis, MO, USA), or 5 ng/ml of TGF‐β (R&D Systems, Minneapolis, MN, USA) as a positive control for 24 hours or 48 hours. Treated LX2 cells were stained with calcein‐AM (Thermo Fisher Scientific) to observe the cell morphology or were collected for RNA extraction. Cell migration was assessed by wound healing. LX2 cells were treated with mitomycin (1 μg/ml) for 2 hours before the scratch. A scratch was performed through the confluent cell monolayer, and detached cells and debris were washed away. Cells were treated with 100 μg/ml of human C1q purified from human serum or 5 ng/ml of TGF‐β for 24 hours. Treated LX2 cells were stained with calcein‐AM for imaging of migrated cells into the wound. Cell viability was measured by the lactate dehydrogenase (LDH) cytotoxicity assay (Dojindo, Japan) using cell‐conditioned media after 48 hours, according to the manufacturer's instructions.

Eguchi A., Iwasa M., Sugimoto R., Tempaku M., Yoshikawa K., Yoshizawa N., Povero D., Sugimoto K., Hasegawa H., Takei Y, & Nakagawa H. (2022). Complement complex 1 subunit q‐mediated hepatic stellate cell activation with connective tissue growth factor elevation is a prognostic factor for survival in rat and human chronic liver diseases. Hepatology Communications, 6(12), 3515-3527.

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Signaling Pathway Activation Analysis

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The following antibodies and reagents were used in this study: anti-phospho-JNK (Cell Signaling 4668); anti-phospho-P38 (Cell Signaling 4511); anti-phospho-ERK (Cell Signaling 4370); anti-phospho-NF-κBp65 (Cell Signaling 3033); anti-phospho-NF-κBp105 (Cell Signaling 3035); anti-phospho-AKT (Ser473; Cell Signaling 4060); anti-JNK (bioworld BS3630); anti-P38 (bioworld BS3567); anti-ERK (bioworld BS1112); anti-NF-κBp65 (Cell Signaling 4764); anti-AKT (bioworld MB0052); anti-TLR2(Cell Signaling 12276); anti-TLR3 (GeneTex GTX113022); anti-TLR4 (GeneTex GTX125909); anti-A20/TNFAIP3 (Cell Signaling 5630); anti-gC1qR/p33 (Millipore MAB1161); Human C1q were obtained from Sigma (C1740); Inhibitors specific for P38 (SB203580), JNK (SP600125), ERK (PD98059), IKKβ (IMD 0354), PI3K (Wortmaninn) and AKT (MK 2206 2HCl) were obtained from selleckchem. CBA Kit was obtained from BD. Ni-NTA Agarose were obtained from QIAGEN. His peptide was obtained from Chinese Peptide Company.

Song X., Yao Z., Yang J., Zhang Z., Deng Y., Li M., Ma C., Yang L., Gao X., Li W., Liu J, & Wei L. (2016). HCV core protein binds to gC1qR to induce A20 expression and inhibit cytokine production through MAPKs and NF-κB signaling pathways. Oncotarget, 7(23), 33796-33808.

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Measuring Autoimmune Antibodies and Immune Complexes

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Blood urea nitrogen concentrations were measured with a Beckman Autoanalyzer (Beckman Coulter, Fullerton, CA). Serum anti-apoferritin IgG antibodies were measured by enzyme-linked immunosorbent assay.^{9 (link)} Briefly, 96-well plates were coated with apoferritin. Serial dilutions of sera were plated and incubated at room temperature for 2 h, followed by HRP-conjugated goat anti-mouse IgG (Kierkegard & Perry Laboratories, Gaithersburg, MD) and OPD peroxidase substrate (Sigma-Aldrich, St. Louis, MO). The OD₄₅₀ was then measured.
Circulating IC levels were measured by C1q-binding enzyme-linked immunosorbent assay, as described previously.^{41 (link)} Briefly, 96-well plates were coated with 10 μg/ml human C1q (Sigma-Aldrich) in carbonate buffer. After blocking with 1% bovine serum albumin, sera samples were loaded in serial dilutions starting at 1/1000, followed by HRP-goat anti-mouse IgG (Sigma, 1:2000) and then TMB (Jackson ImmunoResearch Laboratories, West Grove, PA). The OD₄₅₀ was then measured. Sera samples from unmanipulated wild-type mice were used as negative control.

Alexander J.J., Chaves L.D., Chang A., Jacob A., Ritchie M, & Quigg R.J. (2015). CD11b is protective in complement-mediated immune complex glomerulonephritis. Kidney International, 87(5), 930-939.

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Measuring C1q Binding to Fc Variants

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The interaction of Fc variants with human C1q was measured by ELISA. MaxiSorp 384-well plates (Thermo Fisher Scientific) were directly coated with the anti-CD154 antibodies overnight at 4 °C. After blocking with TBS-T containing 0.5% BSA and 1× Block Ace (DS Pharma Biomedical, Osaka, Japan) for 2 h at room temperature, 3 μg/mL of human C1q (Sigma-Aldrich, St. Louis, MO, USA) was added onto the plates and incubated for 1 h at room temperature. At room temperature, the plates were incubated with sheep anti-human C1q antibody-HRP conjugate (Bio-Rad, Hercules, CA, USA) for 1 h. Washes in PBS-T (pH 7.4) were performed after each subsequent step. TMB substrate (Thermo Fisher Scientific) was subsequently added, and the signal was measured by a plate reader at a wavelength of 450 nm (test wavelength) and 570 nm (reference wavelength).

Sampei Z., Koo C.X., Teo F.J., Toh Y.X., Fukuzawa T., Gan S.W., Nambu T., Ho A., Honda K., Igawa T., Ahmed F., Wang C.I., Fink K, & Nezu J. (2023). Complement Activation by an Anti-Dengue/Zika Antibody with Impaired Fcγ Receptor Binding Provides Strong Efficacy and Abrogates Risk of Antibody-Dependent Enhancement. Antibodies, 12(2), 36.

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C1q Binding to Antibody Isotypes

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The binding of C1q to Ab-1 was examined by using an ELISA based assay. 100 µl 1.5 µg/ml of Ab-1 or hIgG1 isotype (SouthernBiotech) were used to coat the binding surface of a 96-well maxisorb plate (NUNC) overnight at 4°C. The plate was then incubated with a dilution series of human C1q (Sigma-Aldrich) for 2 h, followed by 1-h serial incubations with goat anti-hC1q polyclonal (at dilution 1:1,000; Abcam) and rabbit anti–goat IgG conjugated with alkaline phosphatase (Abcam). The plate was finally washed three times in PBST and once in PBS before the addition of p-nitrophenyl phosphate (Substrate 104; Sigma-Aldrich) at 1 mg/ml (100 µl/well). Absorbance was measured at 405 nm on a PHERAstar plus (BMG Labtech) plate reader after 10–15 min.

Zenonos Z.A., Dummler S.K., Müller-Sienerth N., Chen J., Preiser P.R., Rayner J.C, & Wright G.J. (2015). Basigin is a druggable target for host-oriented antimalarial interventions. The Journal of Experimental Medicine, 212(8), 1145-1151.

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Quantification of Protein-C1q Interactions

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Microtiter wells were coated with 5 μg/mL of human C1q (Merck Calbiochem) in PBS (pH 7.0) and incubated at room temperature for 2 h prior to blocking with 5% skimmed milk for 2 h. The purified proteins, in PBS, were added to wells at a starting concentration of 100 μg/mL and in fivefold dilutions in blocking buffer. Plates were incubated at room temperature for 2 h before being washed with dH₂O + 0.1% Tween‐20. Plates were incubated with detection antiserum (α‐Hu IgGγ₁‐HRP, α‐Mse IgGγ₁‐HRP or α‐Mse IgGγ_2a‐HRP), in blocking buffer, for 2 h. Finally, plates were washed with dH₂O + 0.1% Tween‐20 before being developed with 50 μL of HRP substrate in buffer (Sigmafast OPD tablet and Sigmafast buffer with urea H₂O₂ tablet in 20 mL dH₂O). Absorbance was measured at 450 nm (Tecan Infinite F200 PRO).

Webster G.R., van Dolleweerd C., Guerra T., Stelter S., Hofmann S., Kim M., Teh A.Y., Diogo G.R., Copland A., Paul M.J., Hart P., Reljic R, & Ma J.K. (2018). A polymeric immunoglobulin—antigen fusion protein strategy for enhancing vaccine immunogenicity. Plant Biotechnology Journal, 16(12), 1983-1996.

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C1q Binding Assay for rTs-CRT

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Microtiter plates were coated with different amounts of human C1q (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 µg) (Merck) in 100 μL/well of carbonate buffer (100 mM Na₂CO₃/NaHCO₃, pH 9.6) overnight at 4°C. Control wells were coated with the same amounts of BSA. Following three washes with PBS + 0.05% Tween-20 (PBST), the wells were blocked with 200 µL of 3% BSA in PBS at 37°C for 2 h. After being washed, 0–1.5 µg of rTs-CRT in 100 µL of 20 mM Tris–HCl, pH 7.4, 50 mM NaCl and 1 mM CaCl₂ were added to each well and incubated for 2 h at 37°C. Then, mouse anti-His mAb (1:10,000, TIANGEN) and HRP-conjugated goat anti-mouse IgG (1:10,000) (BD Biosciences, San Jose, CA, USA) were added and incubated for 1 h at 37°C. After the final washing, the substrate o-phenylendiamine dihydrochloride (OPD, Sigma) was added. The absorbance was measured at 450 nm with an ELISA reader (Thermo).

Zhao L., Shao S., Chen Y., Sun X., Sun R., Huang J., Zhan B, & Zhu X. (2017). Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy. Frontiers in Immunology, 8, 636.

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Interaction of rTs-Pmy with C1q

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To further demonstrate the interaction between rTs-Pmy and complement C1q, purified human C1q (5 μg; Merck, Germany) under reducing condition were subjected to SDS-PAGE using 12% polyacrylamide gel followed by either Coomassie bule staining or Far Western blotting. After blocking with 5% dry milk in 1 × PBS, the membrane was incubated with rTs-Pmy (5 μg/ml in PBST and 1% dry milk) at 37°C for 2 h and then washed with PBST. The membrane was probed with an anti-His mAb (rTs-Pmy contains His-tag) (Tiangen, China; 1:5,000 in PBST containing 1% dry milk) for 1 h at room temperature. IRDye 800 CW-labeled goat anti-mouse IgG (LI-COR, Germany; 1:10,000 in PBST containing 1% dry milk) was used as the secondary antibody. Vice versa, rTs-Pmy, non-relevant control BSA and rTs87 (5 μg each) were subjected to 12% SDS-PAGE and then transferred to a NC membrane. After blocking, the membrane was probed with human complement C1q (5 μg/ml in PBST containing 1% dry milk) at 37°C for 2 h. The anti-C1q mAb (Abcam, USA; 1:1,000 in PBST containing 1% dry milk) was used as the primary antibody and IRDye 800 CW-labeled goat anti-mouse IgG (1:10,000 in PBST containing 1% dry milk) was used as the secondary antibody. Membranes were visualized and imaged using an Odyssey infrared imaging system (LI-COR, Germany).

Sun R., Zhao X., Wang Z., Yang J., Zhao L., Zhan B, & Zhu X. (2015). Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation. PLoS Neglected Tropical Diseases, 9(12), e0004310.

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