C9 antibody (Center)

Catalog No. ABIN657704

Product Details anti-C9 Antibody

Target: C9 (Complement Component C9 (C9))

Binding Specificity: AA 191-220, Center

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This C9 antibody is un-conjugated

Application: 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 Details

Application Notes: WB = 1:1000, IHC (p) = 1:10-50

Restrictions: For Research Use only

Independent Validation (WB)

Validation #102069 (Western Blotting)

by: Protein research group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense Denmark

No.: #102069

Date: 01/17/2018

Antigen: C9

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.

Validation Images

Immunoblot using ABIN657704 on human endothelial cell and human astrocytes lysates. C9 protein band intensity is increased in the human endothelial cell after 24h (lane 1-6) compared to 48h (lane 8-13) when treated with nanoparticles. Negative controls are endothelial cells (lane 7) and astrocytes (lane 14) without nanoparticle treatment.

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.

Handling

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 Details for C9

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