mouse monoclonal human clusterin antibody  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: The C7 and clusterin association was confirmed with a native-restricted anti-C7 mAb. (A) The M7-HB2H mAb binding target is located in the C-terminal domains (highlighted in grey) of C7. These domains participate in the formation of the MAC via interaction with the C345C domain of C5, hence, the M7-HB2H antibody would likely bind to an exposed epitope in the native form of C7 (i.e. C7 that is not involved in the assembly of the MAC). The C7 gene contains 18 exons, each exon codes for a specific region of the C7 domain, which corresponds to the exon coding region displayed above. The boundary phase refers to the phase of the corresponding intron. (B) C7 purified from NHS-BioIVT with anti-C7 mAb, M7-HB2H, was separated by SDS-PAGE under non-reducing conditions and the immunoblot was probed with M7-HB2H. Protein composition of the bands visualized in (B) was analzyed by mass spectrometry and protein abundance was quantified for the (C) 100 kDa band and the (D) 75 kDa band. (E) The C7 aa residues detected in the 75 kDa band (3B) are in bold and underlined, along with an illustration of the screened residues in the C7 polypeptide chain highlighted in grey. The screened aa residues are 47% of the total C7 polypeptide chain. Black arrows indicate the exon/intron boundaries. Western blot (B) is representative of at least three independent experiments. Images were taken using Proxima C16 Phi. Figure (A) modified from Massri et al., 2022 .

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Binding Assay, Purification, SDS Page, Western Blot, Mass Spectrometry, Modification

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Clusterin is the most abundant protein detected in the 75 kDa band of purified C7. Protein composition of the bands visualized for C7 purified with pAb P7 ( Figure 2A ) was analzyed by mass spectrometry and protein abundance was quantified for the (A) 100 kDa band and the (B) 75 kDa band. Protein composition of the bands visualized for C7 purified with mAb M7-WU4-15 ( Figure 2B ) was analyzed by mass spectrometry and protein abundance was quantified in the (C) 100 kDa band and the (D) 75 kDa band.

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Purification, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Clusterin purified from NHS with polyclonal and monoclonal antibodies contained low levels of C7. (A) Clusterin purified from NHS-MUI with anti-clusterin pAb, PC, was separated by SDS-PAGE under non-reducing conditions and the immunoblot was probed with the anti-clusterin mAb, MC. The clusterin band on the left was from the 1 st eluate acquired from the antibody-coupled column, while the band on the right was from a 2 nd , consecutive elution step. (B) Coomassie staining of clusterin purified from NHS-MUI with the anti-clusterin pAb, PC, and separated by SDS-PAGE gel under non-reducing conditions. (C) Protein composition of the 75 kDa band visualized in (A) was analyzed by mass spectrometry and protein abundance was quantified. (D) Protein compostion of the 75 kDa band (not shown) from C7 purified with the anti-clusterin mAb, MC-2D5, was analyzed by mass spectrometry and protein abundance was quantified. Results from purified clusterin in (B, C) were acquiered from the 1 st eluate. Western blot and coomassie staining are representative of at least three independent experiments. Images were taken using the ImageQuant™ LAS 400 camera system.

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Purification, SDS Page, Western Blot, Staining, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Western blot analysis reveals strong association between C7 and clusterin. (A, B) Indicated proteins were separated by SDS-PAGE under (A) non-reducing or (B) reducing conditions and immunoblots were probed with the anti-C7 mAb, M7-HB2H. (C, D) Indicated proteins were separated by SDS-PAGE under (C) non-reducing or (D) reducing conditions and immunoblots were probed with the anti-clusterin mAb, MC-2D5. Western blots are representative of at least three independent experiments. Images were taken using Proxima C16 Phi.

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Western Blot, SDS Page

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: C7 and CLU co-elute in high molecular weight complexes. (A) Fractions of pooled NHS (F30-F62) were obtained by size exclusion chromatography and subsequently separated by SDS-PAGE under non-reducing conditions and immunoblots were probed with (B) anti-C7 mAb, M7-HB2H or (C) anti-clusterin mAb, MC-2D5. (D) Summary of (B, C) illustrating the fractions that were either positive or negative for C7 and clusterin. ± indicates fractions where a very faint band was detected due to minute amounts of the protein. (E) Concentration of the C7-CLU complex and native C7 in serum fractions. Note the difference between the y-axis scales, which translates to small concentrations of complexed C7, relative to native C7. NHS, normal human serum.

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Molecular Weight, Size-exclusion Chromatography, SDS Page, Western Blot, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: The C7 and clusterin association was confirmed with a native-restricted anti-C7 mAb. (A) The M7-HB2H mAb binding target is located in the C-terminal domains (highlighted in grey) of C7. These domains participate in the formation of the MAC via interaction with the C345C domain of C5, hence, the M7-HB2H antibody would likely bind to an exposed epitope in the native form of C7 (i.e. C7 that is not involved in the assembly of the MAC). The C7 gene contains 18 exons, each exon codes for a specific region of the C7 domain, which corresponds to the exon coding region displayed above. The boundary phase refers to the phase of the corresponding intron. (B) C7 purified from NHS-BioIVT with anti-C7 mAb, M7-HB2H, was separated by SDS-PAGE under non-reducing conditions and the immunoblot was probed with M7-HB2H. Protein composition of the bands visualized in (B) was analzyed by mass spectrometry and protein abundance was quantified for the (C) 100 kDa band and the (D) 75 kDa band. (E) The C7 aa residues detected in the 75 kDa band (3B) are in bold and underlined, along with an illustration of the screened residues in the C7 polypeptide chain highlighted in grey. The screened aa residues are 47% of the total C7 polypeptide chain. Black arrows indicate the exon/intron boundaries. Western blot (B) is representative of at least three independent experiments. Images were taken using Proxima C16 Phi. Figure (A) modified from Massri et al., 2022 .

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Binding Assay, Purification, SDS Page, Western Blot, Mass Spectrometry, Modification

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Clusterin is the most abundant protein detected in the 75 kDa band of purified C7. Protein composition of the bands visualized for C7 purified with pAb P7 ( Figure 2A ) was analzyed by mass spectrometry and protein abundance was quantified for the (A) 100 kDa band and the (B) 75 kDa band. Protein composition of the bands visualized for C7 purified with mAb M7-WU4-15 ( Figure 2B ) was analyzed by mass spectrometry and protein abundance was quantified in the (C) 100 kDa band and the (D) 75 kDa band.

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Purification, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Clusterin purified from NHS with polyclonal and monoclonal antibodies contained low levels of C7. (A) Clusterin purified from NHS-MUI with anti-clusterin pAb, PC, was separated by SDS-PAGE under non-reducing conditions and the immunoblot was probed with the anti-clusterin mAb, MC. The clusterin band on the left was from the 1 st eluate acquired from the antibody-coupled column, while the band on the right was from a 2 nd , consecutive elution step. (B) Coomassie staining of clusterin purified from NHS-MUI with the anti-clusterin pAb, PC, and separated by SDS-PAGE gel under non-reducing conditions. (C) Protein composition of the 75 kDa band visualized in (A) was analyzed by mass spectrometry and protein abundance was quantified. (D) Protein compostion of the 75 kDa band (not shown) from C7 purified with the anti-clusterin mAb, MC-2D5, was analyzed by mass spectrometry and protein abundance was quantified. Results from purified clusterin in (B, C) were acquiered from the 1 st eluate. Western blot and coomassie staining are representative of at least three independent experiments. Images were taken using the ImageQuant™ LAS 400 camera system.

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Purification, SDS Page, Western Blot, Staining, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Western blot analysis reveals strong association between C7 and clusterin. (A, B) Indicated proteins were separated by SDS-PAGE under (A) non-reducing or (B) reducing conditions and immunoblots were probed with the anti-C7 mAb, M7-HB2H. (C, D) Indicated proteins were separated by SDS-PAGE under (C) non-reducing or (D) reducing conditions and immunoblots were probed with the anti-clusterin mAb, MC-2D5. Western blots are representative of at least three independent experiments. Images were taken using Proxima C16 Phi.

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Western Blot, SDS Page

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: C7 and CLU co-elute in high molecular weight complexes. (A) Fractions of pooled NHS (F30-F62) were obtained by size exclusion chromatography and subsequently separated by SDS-PAGE under non-reducing conditions and immunoblots were probed with (B) anti-C7 mAb, M7-HB2H or (C) anti-clusterin mAb, MC-2D5. (D) Summary of (B, C) illustrating the fractions that were either positive or negative for C7 and clusterin. ± indicates fractions where a very faint band was detected due to minute amounts of the protein. (E) Concentration of the C7-CLU complex and native C7 in serum fractions. Note the difference between the y-axis scales, which translates to small concentrations of complexed C7, relative to native C7. NHS, normal human serum.

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Molecular Weight, Size-exclusion Chromatography, SDS Page, Western Blot, Concentration Assay

Journal: eLife

Article Title: Brain clusterin protein isoforms and mitochondrial localization

doi: 10.7554/eLife.48255

Figure Lengend Snippet: ( A ) Empty vector (pCMV6), V5-tagged pCMV6-human CLU Exon 2–9, or Exon 3–9 constructs were transfected into human SH-SY5Y cells. Cell lysates were harvested and probed for CLU immunoreactivity using anti-V5 (left panel) or anti-Clusterin (right panel). ( B : left panel) pCMV6-human CLU Exon 2–9 or Exon 3–9 constructs were transfected into human SH-SY5Y cells. The resulting cell lysates were treated with PNGase F or Endo H and probed for CLU immunoreactivity using anti-V5. ( B : right panel) Cell lysates harvested from pCMV6-human CLU Exon 3–9-transfected SH-SY5Y cells were treated with O -glycosidase, neuraminidase, or a combination of the two and probed for expression of CLU_45 kDa protein using anti-V5. ( C ) pCMV6-human CLU Exon 3–9-transfected SH-SY5Y cells were subjected to cellular fractionation using the Qiagen Qproteome Mitochondrial Isolation Kit and analyzed for CLU immunoreactivity using anti-V5. Fraction isolation was confirmed using calnexin (ER), Gapdh (cytosol), lamin A/C and lamin B1 (nucleus), and VDAC and Tom20 (mitochondria).

Article Snippet: Primary antibodies used in the studies presented herein include: rabbit polyclonal actin beta (β-actin, Pierce, Cat. #PA5-16914), mouse monoclonal beta-tubulin [TBN06 (Tub 2.5)] (β-tubulin, Pierce, Cat. #MA5-11732), rabbit polyclonal calnexin (H70) (Santa Cruz, Cat. #sc-11397), rabbit monoclonal calreticulin (D3E6) (Cell Signaling, Cat. #12238), rabbit polyclonal clusterin (CLU H-330; Santa Cruz, Cat. #sc-8354), goat polyclonal clusterin-α (CLU M18; Santa Cruz, Cat. #sc-6420), mouse monoclonal clusterin-α (CLU B-5; Santa Cruz, Cat. #sc-5289), mouse monoclonal human clusterin antibody (CLU MAB2937; R and D Systems, Cat. #MAB2937), goat polyclonal human clusterin isoform one antibody (CLU AF2937; R and D Systems, Cat. #AF2937), rabbit monoclonal COX IV (3E11) (Cell Signaling, Cat. #4850), rabbit monoclonal cytochrome C (Cyt.

Techniques: Plasmid Preparation, Construct, Transfection, Expressing, Cell Fractionation, Isolation

Journal: Cell

Article Title: Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease

doi: 10.1016/j.cell.2018.08.067

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-human Clusterin , Bio-Techne , Cat#MAB2937; RRID: AB_2229755.

Techniques: Recombinant, Coagulation, Antibody Labeling, Membrane, Plasmid Preparation, Polymer, Blocking Assay, Staining, Imaging, Software, Hybridization

Journal: Allergy & Rhinology

Article Title: Anti-inflammatory effect of wogonin on allergic responses in ovalbumin-induced allergic rhinitis in the mouse

doi: 10.1177/2152656718764145

Figure Lengend Snippet: Effects of wogonin on the serum levels of cytokines in a mouse model of allergic rhinitis (AR). The concentrations of (A) interleukin (IL) 4, (B) IL-5, and (C) IL-13 in the sera of the mice with AR were measured by using mouse cytokine enzyme-linked immunosorbent assay kits. Independent experiments were performed (n=7). The columns and error bars represent the mean±SE, respectively. *p < 0.05 and ***p < 0.005, significantly different from the OVA group.

Article Snippet: Nasal specimens were incubated with mouse monoclonal antihuman IL-4 antibody (BioVendor Laboratory Medicine Inc., Brno, Czech Republic) and purified antimouse/human IL-5 antibody (BioLegend, San Diego, CA), which was diluted 1:200, for 90 minutes at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Allergy & Rhinology

Article Title: Anti-inflammatory effect of wogonin on allergic responses in ovalbumin-induced allergic rhinitis in the mouse

doi: 10.1177/2152656718764145

Figure Lengend Snippet: Effects of wogonin on cytokine levels in the nasal lavage fluid (NALF) of mice with allergic rhinitis (AR). The concentrations of (A) interleukin (IL) 4, (B) IL-5, (C) IL-13, (D) eotaxin, and (E) RANTES in the NALF of mice with AR were measured by using mouse cytokine enzyme-linked immunosorbent assay kits. Independent experiments were performed (n=7). The columns and error bars represent the mean±SE. *p < 0.05 and **p < 0.005, significantly different from the ovalbumin (OVA) group.

Article Snippet: Nasal specimens were incubated with mouse monoclonal antihuman IL-4 antibody (BioVendor Laboratory Medicine Inc., Brno, Czech Republic) and purified antimouse/human IL-5 antibody (BioLegend, San Diego, CA), which was diluted 1:200, for 90 minutes at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay