Anti-C5a antibody [EPR24303-10]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-C5a antibody [EPR24303-10] (AB281923)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293 (Human embryonic kidney epithelial cell, Left)/ HepG2 (human hepatocellular carcinoma epithelial cell, Right) cells labelling C5a with ab281923 at 1/50 dilution (1ug)/ (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. No expression was observed in cell line : HEK-293.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C5a antibody [EPR24303-10] (AB281923)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling C5a with ab281923 at 1/500 dilution (1.092 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on plasma of human colon. The section was incubated with ab281923 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C5a antibody [EPR24303-10] (AB281923)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labelling C5a with ab281923 at 1/500 dilution (1.092 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human liver. The section was incubated with ab281923 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IP

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Immunoprecipitation - Anti-C5a antibody [EPR24303-10] (AB281923)

C5a was immunoprecipitated from 0.35 mg Human plasma with ab281923 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab281923 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Human plasma 10 μL

Lane 2 : ab281923 IP in Human plasma

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab281923 in human plasma

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 6 seconds.

All lanes:

Immunoprecipitation - Anti-C5a antibody [EPR24303-10] (ab281923)

Observed band size: 110 kDa

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• WB

Lab

Western blot - Anti-C5a antibody [EPR24303-10] (AB281923)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

110 kDa Complement C5 alpha chain is observed. The molecular weight is consistent with what has been described in the literature (PMID : 24071736, 23401592, 25387724).

Very low or no expression of C5 alpha chain is observed in testis, skeletal muscle.

Exposure time : 6 seconds.

All lanes:

Western blot - Anti-C5a antibody [EPR24303-10] (ab281923) at 1/1000 dilution

Lane 1:

Human plasma at 10 µL

Lane 2:

Human testis tissue lysate at 20 µg

Lane 3:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

Observed band size: 110 kDa

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• WB

Lab

Western blot - Anti-C5a antibody [EPR24303-10] (AB281923)

Western blot : Rabbit monoclonal [EPR24303-10] to C5a ab281923 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 180 kDa in Wild-type A549 cell lysates with no signal observed at this size in C5 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-C5a antibody [EPR24303-10] (ab281923) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

C5 knockout A549 at 20 µg

Lane 3:

HepG2 at 20 µg

Lane 4:

U-2 OS at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 188 kDa

Observed band size: 180 kDa

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