Anti-CYR61/CCN1 antibody [EPR20681]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CYR61/CCN1 antibody [EPR20681] (AB230947)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MDA-MB-231 (human breast adenocarcinoma epithelial cell) cells labeling CYR61/CCN1 with ab230947 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in the MDA-MB-231 cell line. Negative control : MCF7 (PMID : 11059746). The nuclear counter stain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CYR61/CCN1 antibody [EPR20681] (AB230947)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized MCF7 (human breast adenocarcinoma epithelial cell, Left) / MDA-MB-231 (human breast adenocarcinoma epithelial cell, Right) cell line labeling CYR61/CCN1with ab230947 at 1/60 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^� 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative control : MCF7 (PMID : 11059746).

• IP

Unknown

Immunoprecipitation - Anti-CYR61/CCN1 antibody [EPR20681] (AB230947)

CYR61/CCN1 was immunoprecipitated from 0.35 mg Saos-2 (human osteosarcoma epithelial) whole cell lysate with ab230947 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab230947 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.
Lane 1 : Saos-2 (human osteosarcoma epithelial) whole cell lysate 10 μg (Input).
Lane 2 : ab230947 IP in Saos-2 whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab230947 in Saos-2 whole cell lysate (-).
Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 8 seconds.

The molecular mass observed is consistent with that of the full-length protein (42 kDa) (PMID : 23798676).

All lanes:

Immunoprecipitation - Anti-CYR61/CCN1 antibody [EPR20681] (ab230947)

Predicted band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-CYR61/CCN1 antibody [EPR20681] (AB230947)

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

The molecular mass observed is consistent with that of the full-length protein (42 kDa) (PMID : 23798676).

All lanes:

Western blot - Anti-CYR61/CCN1 antibody [EPR20681] (ab230947) at 1/5000 dilution

All lanes:

MDA-MB-231 (human breast adenocarcinoma 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: 42 kDa

Observed band size: 42 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-CYR61/CCN1 antibody [EPR20681] (AB230947)

Lanes 1-4 : Merged signal (red and green). Green - ab230947 observed at 47 kDa. Red - loading control ab8245 observed at 36 kDa.

ab230947 Anti-CYR61/CCN1 antibody [EPR20681] was shown to specifically react with CYR61/CCN1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265288 (knockout cell lysate ab257406) was used. Wild-type and CYR61/CCN1 knockout samples were subjected to SDS-PAGE. ab230947 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CYR61/CCN1 antibody [EPR20681] (ab230947) at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CYR61 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CYR61 (CCN1) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cyr61-ccn1-knockout-hela-cell-line-ab265288'>ab265288</a>)

Lane 3:

MDA-MB-231 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 42 kDa

Observed band size: 47 kDa

false

• WB

Supplier Data

Western blot - Anti-CYR61/CCN1 antibody [EPR20681] (AB230947)

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

The expression profile observed is consistent with the literature, with the 42 kDa band being the full-length protein and the 19 kDa band likely a degradation product (PMID : 23798676; PMID : 16266990; PMID : 8657105).

Negative control : MCF7 (PMID 11059746).

All lanes:

Western blot - Anti-CYR61/CCN1 antibody [EPR20681] (ab230947) at 1/1000 dilution

Lane 1:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Saos-2 (human osteosarcoma epithelial) whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma 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: 42 kDa

false

Exposure time: 3min