All lanes : Anti-CYR61/CCN1 antibody [EPR20681] (ab230947) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CYR61 knockout HeLa cell lysate
Lane 3 : MDA-MB-231 cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 42 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?

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.

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.

Anti-CYR61/CCN1 antibody [EPR20681] (ab230947) at 1/5000 dilution + MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa


Exposure time: 3 minutes

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

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

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).

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/CCN1 with 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).

All lanes : Anti-CYR61/CCN1 antibody [EPR20681] (ab230947) at 1/1000 dilution

Lane 1 : PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : Saos-2 (human osteosarcoma epithelial) whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 42 kDa


Exposure time: 3 minutes

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).