Anti-Cyclophilin A antibody [1F4-1B5]

• WB

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Western blot - Anti-Cyclophilin A antibody [1F4-1B5] (AB58144)

Cyclophilin A antibody (ab58144) at 1ug/lane + Jurkat cell lysate at 25ug/lane.

This image was generated using the ascites version of the product.

All lanes:

Western blot - Anti-Cyclophilin A antibody [1F4-1B5] (ab58144)

Predicted band size: 18 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin A antibody [1F4-1B5] (AB58144)

ICC/IF image of ab58144 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab58144, 1μg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

This image was generated using the ascites version of the product.

• Flow Cyt

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Flow Cytometry - Anti-Cyclophilin A antibody [1F4-1B5] (AB58144)

Overlay histogram showing HeLa cells stained with ab58144 (red line). The cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab58144, 0.5μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This image was generated using the ascites version of the product.

• IP

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Immunoprecipitation - Anti-Cyclophilin A antibody [1F4-1B5] (AB58144)

Cyclophilin A was immunoprecipitated using 0.5mg Hela whole cell extract, 10μg of Mouse monoclonal to Cyclophilin A 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 ab58144.
Secondary : Protein G-HRP at 1/500 dilution.
Band : 18kDa : Cyclophilin A.

This image was generated using the ascites version of the product.

All lanes:

Immunoprecipitation - Anti-Cyclophilin A antibody [1F4-1B5] (ab58144)

Predicted band size: 18 kDa

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