Anti-C3 antibody [EPR26515-77]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-C3 antibody [EPR26515-77] (AB321966)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% TritonX-100 permeabilized J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage) cells labelling C3 with ab321966 at 1/50 (9.72 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing weak cytoplasmic staining in J774A.1 cell line and the expression increased after treatment with LPS at 1ug/ml for 24 hours then adding BFA at 300ng/ml for 24 hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• WB

Lab

Western blot - Anti-C3 antibody [EPR26515-77] (AB321966)

Western blot : Anti-C3 antibody [EPR26515-77] ab321966 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 170 kDa in Wild-type 3T3-L1 after treatment with brefeldin A, with no signal observed at this size in C3 knockout 3T3-L1 samples. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-C3 antibody [EPR26515-77] (ab321966) at 1/1000 dilution

Lane 1:

Wild-type 3T3-L1 Treated: Brefeldin A (300 ng/mL, 24 h) cell lysates at 20 µg

Lane 2:

Wild-type 3T3-L1 Vehicle Control for Brefeldin A (0 ng/mL, 24 h) cell lysates at 20 µg

Lane 3:

C3 knockout 3T3-L1: Treated: Brefeldin A (300 ng/mL, 24 h) cell lysates at 20 µg

Lane 4:

C3 knockout 3T3-L1 Vehicle Control for Brefeldin A (0 ng/mL, 24 h) cell lysates at 20 µg

Secondary

All lanes:

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

Predicted band size: 187 kDa

Observed band size: 170 kDa

false

• WB

Supplier Data

Western blot - Anti-C3 antibody [EPR26515-77] (AB321966)

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

The band around 186 kDa is the Complement C3 and 110 kDa the Complement C3 alpha chain.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lanes 1-2 : 92 seconds; Lane 3 : 158 seconds.

All lanes:

Western blot - Anti-C3 antibody [EPR26515-77] (ab321966) at 1/1000 dilution

Lane 1:

3T3-L1 (mouse embryonic fibroblast) transfected with scrambled siRNA control whole cell lysate at 30 µg

Lane 2:

3T3-L1 transfected with siRNA specifically targeting C3 whole cell lysate at 30 µg

Lane 3:

Hepa1-6 (mouse hepatoma epithelial cell) whole cell lysate at 30 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 186 kDa,110 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-C3 antibody [EPR26515-77] (AB321966)

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

The band around 110 kDa is the Complement C3 alpha chain and 38 kDa the Complement C3c alpha' chain fragment 2.

All lanes:

Western blot - Anti-C3 antibody [EPR26515-77] (ab321966) at 1/1000 dilution

Lane 1:

Mouse liver tissue whole cell lysate at 30 µg

Lane 2:

Mouse brain tissue whole cell lysate at 30 µg

Lane 3:

Mouse kidney tissue whole cell lysate at 30 µg

Lane 4:

Mouse testis tissue whole cell lysate at 30 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 110 kDa,38 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-C3 antibody [EPR26515-77] (AB321966)

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

The band around 186 kDa is the Complement C3 and 110 kDa the Complement C3 alpha chain.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lanes 1-2 : 6 seconds; Lanes 3-5 : 180 seconds.

All lanes:

Western blot - Anti-C3 antibody [EPR26515-77] (ab321966) at 1/1000 dilution

Lane 1:

Untreated J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage ) whole cell lysate at 30 µg

Lane 2:

J774A.1 treated with 1ug/ml LPS(Lipopolysaccharide) and 300 ng/ml BFA(Brefeldin A) for 24 hours whole cell lysate at 30 µg

Lane 3:

Untreated RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 30 µg

Lane 4:

RAW 264.7 treated with 1ug/ml LPS(Lipopolysaccharide) for 24 hours whole cell lysate at 30 µg

Lane 5:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 30 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 186 kDa,36 kDa,110 kDa

false