Recombinant Anti-Cyclophilin F antibody [EPR11311-121] (ab231155)

ab231155 was shown to specifically react with Cyclophilin F in wild-type HAP1 cells as signal was lost in Cyclophilin F knockout cells. Wild-type and Cyclophilin F knockout samples were subjected to SDS-PAGE. Ab231155 and ab181602 (Human anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/200000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrumentusing the ECL technique.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Cyclophilin F with ab231155 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 (green). Confocal image showing Cyclophilin F staining and mitochondrial localization in the HeLa cell line. The nuclear counter stain is DAPI (blue).

Mitochondria are stained with ab33985 Anti-COX IV (mouse mAb) - Mitochondrial Marker followed by ab150120 AlexaFluor^®594 Goat anti-Mouse secondary both at 1/1000 dilution (red).

-ve control 1: ab231155 (Rabbit anti-Cyclophilin F) at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab33985 Anti-COX IV (mouse mAb) - Mitochondrial Marker at 1/1000 dilution followed by ab150077 (AlexaFluor®488 Goat anti-Rabbit secondary) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Cyclophilin F with ab231155 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Granular cytoplasmic staining in mouse colon (PMID: 23991223, PMID: 15344907, PMID: 26258444) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Jurkat (human T cell leukemia T lymphocyte) cell line labeling Cyclophilin F with ab231155 at 1/600 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells incubated with secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

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

Exposure times:

Lanes 1-5: 59 seconds

Lane 6: 37 seconds

Lane 7-8:3 minutes 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Wildtype and Cyclophilin F knockout HAP1 (human chronic myelogenous leukemia near-haploid cell line) cells labeling Cyclophilin F with ab231155 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 (green). Confocal image showing Cyclophilin F staining and mitochondrial co-localization in wildtype HAP1 cells, with loss of the signal in the Cyclophin F-knockout HAP1 cells. The nuclear counter stain is DAPI (blue).

Mitochondria are stained with with ab33985 Anti-COX IV (mouse mAb) - Mitochondrial Marker followed by ab150120 AlexaFluor^®594 Goat anti-Mouse secondary both at 1/1000 dilution (red).

-ve control 1: ab231155 (Rabbit anti-Cyclophilin F) at 1/1000 dilution followed by ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab33985 Anti-COX IV (mouse mAb) - Mitochondrial Marker at 1/1000 dilution followed by ab150077 (AlexaFluor^®488 Goat anti-Rabbit secondary) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human colonic carcinoma tissue labeling Cyclophilin F with ab231155 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Granular cytoplasmic staining in tumor cells of human colon carcinoma (PMID: 23991223, PMID: 15344907, PMID: 26258444) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Cyclophilin F with ab231155 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Granular cytoplasmic staining in human kidney (PMID: 23991223, PMID: 15344907, PMID: 26258444) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling Cyclophilin F with ab231155 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Granular cytoplasmic staining in human pancreas (PMID: 23991223, PMID: 15344907, PMID: 26258444) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling Cyclophilin F with ab231155 at 1/600 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells incubated with secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.