Anti-CTGF antibody [EPR20728]

• IP

Unknown

Immunoprecipitation - Anti-CTGF antibody [EPR20728] (AB209780)

CTGF was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab209780 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab209780 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.
Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 μg (Input).
Lane 2 : ab209780 IP in HepG2 whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab209780 in HepG2 whole cell lysate (-).
Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 minutes.

The molecular mass observed is consistent with the literature. (PMID : 27126736)

All lanes:

Immunoprecipitation - Anti-CTGF antibody [EPR20728] (ab209780)

Predicted band size: 38 kDa

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CTGF antibody [EPR20728] (AB209780)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% TritonX-100 permeabilized Rat-1 (rat embryonic fibroblast) cells labelling CTGF with ab209780 at 1/100 (21.45 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green).

Confocal image showing increased cytoplasmic staining in Rat-1 cells (shown in green) treated with TGFβ (10 ng/mL) and Herparin sodium salt (50 μg/mL) for 24 hr. The counterstain was observed in magenta. Nuclear DNA was labeled 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 (2.5 ug/ml) dilution (Magenta).

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CTGF antibody [EPR20728] (AB209780)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling CTGF with ab209780 at 1/100 (21.45 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green).

Confocal image showing increased cytoplasmic staining in NIH/3T3 cells (shown in green) treated with TGFβ (10 ng/mL) and Herparin sodium salt (50 μg/mL) for 24 hr. The counterstain was observed in magenta. Nuclear DNA was labeled 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).

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

• WB

Lab

Western blot - Anti-CTGF antibody [EPR20728] (AB209780)

False colour image of Western blot : Anti-CTGF antibody [EPR20728] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab209780 was shown to bind specifically to CTGF. A band was observed at 38 kDa in wild-type U-2 OS cell lysates with no signal observed at this size in CTGF knockout cell line ab261880 (knockout cell lysate ab261689). To generate this image, wild-type and CTGF knockout U-2 OS cell lysates were analysed. First, 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 IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-CTGF antibody [EPR20728] (ab209780) at 1/1000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

Empty

Lane 3:

CTGF knockout U-2 OS cell lysate at 20 µg

Lane 5:

HepG2 cell lysate at 20 µg

Predicted band size: 38 kDa

Observed band size: 38 kDa

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• WB

Unknown

Western blot - Anti-CTGF antibody [EPR20728] (AB209780)

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

The level of CTGF expression can be induced by TGF beta treatment (PMID : 17786299).

CTGF is constitutively expressed in HepG2 cells (PMID : 15886528).

All lanes:

Western blot - Anti-CTGF antibody [EPR20728] (ab209780) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) starved for 18 hours, whole cell lysate at 20 µg

Lane 2:

NIH/3T3 starved for 18 hours, then treated with 10 ng/ml transforming growth factor-β (TGF-β1, <a href='/en-us/products/proteins-peptides/recombinant-human-tgf-beta-1-protein-active-ab50036'>ab50036</a>) and 50 µg/ml Heparin sodium salt for 24 hours, whole cell lysate at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 38 kDa,44 kDa

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