All lanes : Anti-Cyclophilin B antibody (ab16045) at 1/5000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PPIB knockout HAP1 whole cell lysate
Lane 3 : Jurkat whole cell lysate
Lane 4 : U87-MG whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 21 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab16045 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab16045 was shown to specifically react with PPIB in wild-type HAP1 cells as signal was lost in PPIB knockout cells. Wild-type and PPIB knockout samples were subjected to SDS-PAGE. Ab16045 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution 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.

ab16045 stained HeLa cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab16045 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

All lanes : Anti-Cyclophilin B antibody (ab16045) at 1 µg/ml

Lane 1 : Rat Liver
Lane 2 : Mouse 3T3
Lane 3 : Dog
Lane 4 : Chicken

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

All lanes : Anti-Cyclophilin B antibody (ab16045) at 1 µg/ml

Lane 1 : HeLa nuclear lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : A431 whole cell lysate
Lane 4 : Jurkat whole cell lysate
Lane 5 : HEK293 whole cell lysate
Lane 6 : HeLa nuclear lysate with Human Cyclophilin B peptide (ab16277) at 1 µg/ml
Lane 7 : HeLa whole cell lysate with Human Cyclophilin B peptide (ab16277) at 1 µg/ml
Lane 8 : A431 whole cell lysate with Human Cyclophilin B peptide (ab16277) at 1 µg/ml
Lane 9 : Jurkat whole cell lysate with Human Cyclophilin B peptide (ab16277) at 1 µg
Lane 10 : HEK293 whole cell lysate with Human Cyclophilin B peptide (ab16277) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 21 kDa


Exposure time: 30 seconds

Cyclophilin B was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Cyclophilin B and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab16045.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 21kDa: Cyclophilin B.

ICC/IF image of ab16045 stained NIH/3T3 cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab16045, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

ab16045 (1/1000) staining Cyclophilin B in assynchronous HeLa cells (green). Cells were fixed with Paraformaldehyde and counter-stained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.

See Abreview

All lanes : Anti-Cyclophilin B antibody (ab16045) at 1/1000 dilution

Lane 1 : Whole cell lysate prepared from rat pancreatic AR42J cells, which were treated with 10nM dexamethasone for 48 hours.
Lane 2 : Whole cell lysate for negative control, prepared from rat pancreatic AR42J cells (specific knock down of cyclophilin B/PpiB by siRNA), which were treated with 10nM dexamethasone for 48 hours.

Secondary
All lanes : Goat-anti-Rabbit HRP-conjugated polyclonal at 1/2000 dilution

Developed using the ECL technique.

Predicted band size: 21 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute


Primary antibody incubated for 12 hours at 4°C.
Blocking step performed using 5% milk, 1 hour at 20°C.

See Abreview

Anti-Cyclophilin B antibody (ab16045) at 0.5 µg/ml + Recombinant Human Cyclophilin B protein (ab88801) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 21 kDa


Exposure time: 30 seconds