All lanes : Anti-CIRP antibody [EPR18783] (ab191885) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CIRP knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 19 kDa

Lanes 1-2: Merged signal (red and green). Green - ab191885 observed at 19 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab191885 Anti-CIRP antibody [EPR18783] was shown to specifically react with CIRP in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266187 (knockout cell lysate ab257252) was used. Wild-type and CIRP knockout samples were subjected to SDS-PAGE. ab191885 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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.

 

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling CIRP using ab191885 at 1/1000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counterstain: Hematoxylin.
Inset image: negative control obtained using PBS instead of ab191885, and secondary antibody only.
Note: Nuclear staining on normal human endometrium.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling CIRP with ab191885 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:
1. ab191885 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

All lanes : Anti-CIRP antibody [EPR18783] (ab191885) at 1/1000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 19 kDa
Observed band size: 19 kDa


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

All lanes : Anti-CIRP antibody [EPR18783] (ab191885) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated at 37°C for 48 hours
Lane 2 : HeLa whole cell lysate treated at 32°C for 48 hours
Lane 3 : HCT 116 (Human colorectal carcinoma cell line) whole cell lysate
Lane 4 : HepG2 (Human liver hepatocellular carcinoma) 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: 19 kDa
Observed band size: 19 kDa


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

The expression profile observed is consistent with what has been described in the literature (PMID:20735994)

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling CIRP using ab191885 at 1/1000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counterstain: Hematoxylin.
Inset image: negative control obtained using PBS instead of ab191885, and secondary antibody only.
Note: Nuclear and weak cytoplasm staining on tumor cells of breast cancer.
Mol Carcinog. 2010 49, 130–140.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (Human colorectal carcinoma cell line) cells labeling CIRP with ab191885 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HCT 116 cell line.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:
1. ab191885 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Immunoprecipitation of CIRP from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate achieved using ab191885 at 1/20 dilution.
Lane 1: Input: 10µg of HeLa whole cell lysate.
Lane 2: HeLa whole cell lysate following IP with ab191885.
Lane 3: negative control: IP using Rabbit monoclonal IgG (ab172730) instead of ab191885 in HeLa whole cell lysates.
Western blot was performed using ab191885 at 1/1000 dilution.
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/10000 was used for detection.
Blocking and dilution buffer and concentration: 5% NFDM/TBST. 3 second exposure.