Anti-Cyclophilin B antibody - Loading Control

9 product images

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  Immunoprecipitation - Anti-Cyclophilin B antibody - Loading Control (ab16045)

  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.

  All lanes: Immunoprecipitation - Anti-Cyclophilin B antibody - Loading Control (ab16045)

  Predicted band size: 24 kDa

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  Western blot - Anti-Cyclophilin B antibody - Loading Control (ab16045)

  Cyclophilin B Western blot staining using rabbit Anti-Cyclophilin B antibody

  Lanes 1 - 4: Merged signal (red and green). Green - ab16045 observed at 24 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

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

  Lane 1: Wild-type HAP1 whole cell lysate at 20 µg

  Lane 2: PPIB knockout HAP1 whole cell lysate at 20 µg

  Lane 3: Jurkat whole cell lysate at 20 µg

  Lane 4: U87-MG whole cell lysate at 20 µg

  Predicted band size: 24 kDa

  Observed band size: 24 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin B antibody (ab16045)

  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 (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.

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  Western blot - Anti-Cyclophilin B antibody - Loading Control (ab16045)

  Cyclophilin B Western blot staining using rabbit Anti-Cyclophilin B antibody

  All lanes: Western blot - Anti-Cyclophilin B antibody - Loading Control (ab16045) at 1 µg/mL

  Lane 1: Rat Liver at 20 µg

  Lane 2: Mouse 3T3 at 20 µg

  Lane 3: Dog at 20 µg

  Lane 4: Chicken at 20 µg

  Secondary

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

  Performed under reducing conditions.

  Predicted band size: 24 kDa

  Observed band size: 25 kDa

  Exposure time: 30s

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  Western blot - Anti-Cyclophilin B antibody - Loading Control (ab16045)

  Cyclophilin B Western blot staining using rabbit Anti-Cyclophilin B antibody

  All lanes: Western blot - Anti-Cyclophilin B antibody - Loading Control (ab16045) at 1 µg/mL

  Lane 1: HeLa nuclear lysate at 20 µg

  Lane 2: HeLa whole cell lysate at 20 µg

  Lane 3: A431 whole cell lysate at 20 µg

  Lane 4: Jurkat whole cell lysate at 20 µg

  Lane 5: HEK293 whole cell lysate at 20 µg

  Lane 6: HeLa nuclear lysate at 20 µg with Human Cyclophilin B peptide (ab16277)

  Lane 7: HeLa whole cell lysate at 20 µg with Human Cyclophilin B peptide (ab16277)

  Lane 8: A431 whole cell lysate at 20 µg with Human Cyclophilin B peptide (ab16277)

  Lane 9: Jurkat whole cell lysate at 20 µg with Human Cyclophilin B peptide (ab16277)

  Lane 10: HEK293 whole cell lysate at 20 µg with Human Cyclophilin B peptide (ab16277)

  Secondary

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

  Performed under reducing conditions.

  Predicted band size: 24 kDa

  Observed band size: 21 kDa

  Exposure time: 30s

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  This image is courtesy of a customer review submitted by Dr Kirk McManus

  Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin B antibody (ab16045)

  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.

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  Image courtesy of Dr M Schrader by customer review.

  Western blot - Anti-Cyclophilin B antibody - Loading Control (ab16045)

  Cyclophilin B Western blot staining using rabbit Anti-Cyclophilin B antibody

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

  All lanes: Western blot - Anti-Cyclophilin B antibody - Loading Control (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: 24 kDa

  Observed band size: 23 kDa

  Exposure time: 1min

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  Western blot - Anti-Cyclophilin B antibody - Loading Control (ab16045)

  Cyclophilin B Western blot staining using rabbit Anti-Cyclophilin B antibody

  All lanes: Western blot - Anti-Cyclophilin B antibody - Loading Control (ab16045) at 0.5 µg/mL

  All lanes: Recombinant Human Cyclophilin B protein (ab88801) at 0.01 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (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: 24 kDa

  Exposure time: 30s

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  Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin B antibody (ab16045)

  ab16045 staining Cyclophilin B in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab16045 at 1µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.