Anti-Cyclophilin F抗体[E11AE12BD4] (ab110324)

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Cyclophilin F knockout  HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: Hek293 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab110324 observed at 24 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab110324 detected the expected band for Cyclophilin F in wild type HAP1 cells and the band was not seen in Cyclophilin F knockout HAP1 cells. Wild-type and Cyclophilin F knockout samples were subjected to SDS-PAGE.  Ab110324 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry analysis using ab110324 at 1µg/ml staining Cyclophilin 40 in Cultured Human embryonic lung-derived fibroblasts (strain MRC5), (fixed, treated for heat-induced antigen retrieval, permeabilized) followed by an AlexaFluor® 488-conjugated-goat-anti-mouse IgG1 isotype specific secondary antibody (2 µg/ml).

Immunohistological analysis using ab110324 at 1µg/ml staining Cyclophilin F in Human cerebellum tissue (Formalin-fixed, Paraffin-embedded).
Note: immunoactivity is most intense in neuronal cell bodies, most notably in the large Purkinje cells.

All lanes : Anti-Cyclophilin F antibody [E11AE12BD4] (ab110324) at 1/10000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 22 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab110324 observed at 23 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) observed at 37 kDa.

 ab110324 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. ab110324 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Cyclophilin F antibody [E11AE12BD4] (ab110324) at 1/1000 dilution

Lane 1 : WT mouse liver mitochondria lysate at 25 µg
Lane 2 : CypD KO mouse liver mitochondria lysate at 25 µg
Lane 3 : WT mouse liver mitochondria lysate at 35 µg
Lane 4 : CypD KO mouse liver mitochondria lysate at 35 µg
Lane 5 : WT mouse liver mitochondria lysate at 50 µg
Lane 6 : CypD KO mouse liver mitochondria lysate at 50 µg

Secondary
All lanes : HRP-conjugated goat anti-mouse IgG polyclonal at 1/4000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 22 kDa
Observed band size: 20 kDa why is the actual band size different from the predicted?
Additional bands at: 13 kDa (possible non-specific binding)


Exposure time: 5 seconds

Blocked with 5% milk for 1 hour at 25°C.

Incubated with the primary antibody diluted in PBS-T + 5% milk for 16 hours at 4°C.

See Abreview

All lanes : Anti-Cyclophilin F antibody [E11AE12BD4] (ab110324) at 1 µg/ml

Lane 1 : Isolated mitochondria from Human heart at 5 µg
Lane 2 : Isolated mitochondria from Bovine heart at 1 µg
Lane 3 : Isolated mitochondria from Rat heart at 10 µg
Lane 4 : Isolated mitochondria from Mouse heart at 10 µg
Lane 5 : Isolated mitochondria from HepG2 cells at 20 µg

Predicted band size: 22 kDa

Flow cytometric analysis using ab110324 at 1µg/ml staining Cyclophilin F in HL60 cells (blue). Isotype control antibody (red).