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C9 antibody (Center)

C9 Reactivity: Human WB, IHC (p) Host: Rabbit Polyclonal RB33918 unconjugated
Catalog No. ABIN657704
  • Target See all C9 Antibodies
    C9 (Complement Component C9 (C9))
    Binding Specificity
    • 16
    • 8
    • 6
    • 4
    • 3
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    AA 191-220, Center
    Reactivity
    • 57
    • 3
    • 2
    • 2
    • 2
    • 2
    Human
    Host
    • 48
    • 11
    • 1
    • 1
    Rabbit
    Clonality
    • 49
    • 11
    Polyclonal
    Conjugate
    • 28
    • 8
    • 4
    • 3
    • 3
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    This C9 antibody is un-conjugated
    Application
    • 46
    • 24
    • 23
    • 21
    • 12
    • 10
    • 9
    • 7
    • 7
    • 6
    • 6
    • 2
    • 2
    • 1
    Western Blotting (WB), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p))
    Specificity
    This C9 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 190-220 amino acids from the Central region of human C9.
    Purification
    This antibody is purified through a protein A column, followed by peptide affinity purification.
    Immunogen
    This C9 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 191-220 AA from the Central region of human C9.
    Clone
    RB33918
    Isotype
    Ig Fraction
  • Application Notes
    WB = 1:1000, IHC (p) = 1:10-50
    Restrictions
    For Research Use only
  • Validation #102069 (Western Blotting)
    'Independent Validation' Badge
    by
    Protein research group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense Denmark
    No.
    #102069
    Date
    01/17/2018
    Antigen
    C9
    Lot Number
    Method validated
    Western Blotting
    Positive Control
    Human endothelial cells with nanoparticle treatment
    Negative Control
    Human endothelial cells and astrocytes without nanoparticle treatment
    Notes

    Passed. ABIN657704 specifically recognizes C9 in human endothelial cells.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN657704
    Secondary Antibody
    anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074S)
    Full Protocol
    • Grow human endothelial cells and astrocytes in EndoGRO-MV Complete Media kit (Millipore, SCME004, lot 2885245) supplemented with serum (Millipore, lot 1000958) at 37°C and 5% CO2 in 8ml on a petri dish to 80-90 % confluency.
    • Add PVP coated silver nanoparticles (50nm Silver Nanospheres, Nanocomposix USA lot no JRC0234) to the endothelial cells for 24h and 48h.
    • Lyse 106 cells in 100µl per well cold lysis buffer (6M Urea, 2M Thiourea, 10mM DTT).
    • Determine total protein content of the lysates using Qubit Protein Assay Kit (ThermoFisher Scientific, Q33211).
    • Denature 20µg of total protein for 5min at 95°C in 5µl Pierce LDS Sample Buffer, Non-Reducing (Thermo Scientific, b31010, lot 151109002) and separate proteins in an XCell SureLock Mini-Cell (Life Technologies, EI0001, lot 007469934) in a Bolt 4-12% Bis-Tris Plus gel (Thermo scientific, NW04125BOX, lot 17120471) for 60min at 100V.
    • Transfer proteins onto PVDF membrane (Sigma-Aldrich, IPFL00005) with a Western blotting system for 20min at 18V.
    • Block the membrane with TBST and 5% non-fat milk powder (Sigma-Aldrich, 70166-500G, lot SZBF3420V) for 1h at RT.
    • Incubation with primary rabbit anti-C9 antibody (antibodies-online, ABIN657704, lot SA110711AL) diluted 1:1000 TBST and 5% non-fat milk powder for ON at 4°C.
    • Wash membrane 3x for 15min with TBST.
    • Incubation with secondary anti-rabbit IgG HRP-linked antibody (Cell Signaling, 7074S) diluted 1:10000 in TBST and 5% non-fat milk powder for 2h at RT.
    • Wash membrane 3x for 15min with TBS.
    • Reveal protein bands using Luminata Forte Western HRP Substrate (Millipore, WBLUF0100) on a UVP multispectral imaging system (AH diagnostics, Denmark).
    • Incubate the membrane in Restore Plus Western blot Stripping Buffer (Thermo Scientific, 46430, lot RL241559) for 20min at RT.
    • Wash membrane 3x for 15min with TBST.
    • Incubation with rabbit anti-GAPDH antibody (Cell Signaling Technology, 2118) diluted 1:5000 in TBST and 5% non-fat milk powder for 2h at RT.
    • Wash membrane 3x for 15min with TBS.
    • Reveal protein bands as described above.
    Experimental Notes

    Upon addition of the nanoparticles, the endothelial cells’ proteome revealed high expression of C9 at the 24h time point and reduced expression at the 48h time point. We validated the proteomics results by western blotting and we confirm that the C9 antibody ABIN657704 reveals a protein of the expected molecular weight (60kDa) in lysates of endothelial cells. The protein band is only visible in the nanoparticle treated cells but not the negative controls.

  • Format
    Liquid
    Concentration
    0.36 mg/mL
    Buffer
    PBS with 0.09 % (W/V) sodium azide
    Preservative
    Sodium azide
    Precaution of Use
    This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Storage
    4 °C/-20 °C
    Storage Comment
    C9 Antibody (Center) can be refrigerated at 2-8 °C for up to 6 months. For long term storage, place the at -20 °C.
    Expiry Date
    6 months
  • Target
    C9 (Complement Component C9 (C9))
    Alternative Name
    C9 (C9 Products)
    Synonyms
    LOC100037951 antibody, LOC100136130 antibody, c9 antibody, wu:fb60b05 antibody, wu:fd50e04 antibody, zgc:112272 antibody, complement C9 antibody, complement component 9 antibody, complement component 9 L homeolog antibody, C9 antibody, c9 antibody, c9.L antibody
    Background
    This gene encodes the final component of the complement system. It participates in the formation of the Membrane Attack Complex (MAC). The MAC assembles on bacterial membranes to form a pore, permitting disruption of bacterial membrane organization. Mutations in this gene cause component C9 deficiency. [provided by RefSeq].
    Synonyms: Complement component C9,C9,
    Molecular Weight
    63173 DA
    Gene ID
    735
    NCBI Accession
    NP_001728
    UniProt
    P02748
    Pathways
    Complement System
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