Anti-Cyclophilin F antibody [EPR11311-121]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

Intracellular 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

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 Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

Intracellular 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 (cellsincubated with secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^� 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

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).

• WB

Unknown

Western blot - 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.

All lanes:

Western blot - Anti-Cyclophilin F antibody [EPR11311-121] (ab231155) at 1/1000 dilution

Lane 1:

Wild type HAP1 whole cell lysate at 20 µg

Lane 2:

Cyclophilin F knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HEK-293 (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix 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: 22 kDa

Observed band size: 18 kDa

false

Exposure time: 59s

• WB

Lab

Western blot - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

Lanes 1- 2 : Merged signal (red and green). Green - ab231155 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab231155 was shown to react with Cyclophilin F in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266077 (knockout cell lysate ab257039) was used. Wild-type HEK-293T and PPIF knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab231155 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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-Cyclophilin F antibody [EPR11311-121] (ab231155) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

PPIF knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human PPIF (Cyclophilin F) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-ppif-cyclophilin-f-knockout-hek-293t-cell-line-ab266077'>ab266077</a>)

Predicted band size: 22 kDa

Observed band size: 23 kDa

false

• WB

Unknown

Western blot - Anti-Cyclophilin F antibody [EPR11311-121] (AB231155)

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

Exposure times :

Lanes 1-5 : 59 seconds

Lane 6 : 37 seconds

Lane 7-8 : 3 minutes

All lanes:

Anti-Thrombospondin antibody (<a href='/en-us/products/unavailable/thrombospondin-antibody-ab231115'>ab231115</a>) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 4:

Human liver lysate at 20 µg

Lane 5:

Human heart lysate at 20 µg

Lane 6:

Mouse heart lysate at 20 µg

Lane 7:

Rat liver lysate at 20 µg

Lane 8:

Rat heart 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: 22 kDa

Observed band size: 18 kDa

false