Anti-Cyclophilin B antibody - Loading Control

• WB

Lab

Western blot - Anti-Cyclophilin B antibody - Loading Control (AB16045)

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.

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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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin B antibody - Loading Control (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 ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and 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.

• ICC/IF

AbReview7657****

Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin B antibody - Loading Control (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.

This image is courtesy of an Abreview submitted by Dr Kirk McManus

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cyclophilin B antibody - Loading Control (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 (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.

• IP

Unknown

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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• WB

AbReview20583****

Western blot - Anti-Cyclophilin B antibody - Loading Control (AB16045)

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

Predicted band size: 24 kDa

Observed band size: 23 kDa

true

Exposure time: 1min

Image courtesy of Dr M Schrader by Abreview.

• WB

Unknown

Western blot - Anti-Cyclophilin B antibody - Loading Control (AB16045)

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 (<a href='/en-us/products/unavailable/human-cyclophilin-b-peptide-ab16277'>ab16277</a>)

Lane 7:

HeLa whole cell lysate at 20 µg with Human Cyclophilin B peptide (<a href='/en-us/products/unavailable/human-cyclophilin-b-peptide-ab16277'>ab16277</a>)

Lane 8:

A431 whole cell lysate at 20 µg with Human Cyclophilin B peptide (<a href='/en-us/products/unavailable/human-cyclophilin-b-peptide-ab16277'>ab16277</a>)

Lane 9:

Jurkat whole cell lysate at 20 µg with Human Cyclophilin B peptide (<a href='/en-us/products/unavailable/human-cyclophilin-b-peptide-ab16277'>ab16277</a>)

Lane 10:

HEK293 whole cell lysate at 20 µg with Human Cyclophilin B peptide (<a href='/en-us/products/unavailable/human-cyclophilin-b-peptide-ab16277'>ab16277</a>)

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-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 24 kDa

Observed band size: 21 kDa

false

Exposure time: 30s

• WB

Unknown

Western blot - Anti-Cyclophilin B antibody - Loading Control (AB16045)

All lanes:

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

All lanes:

Recombinant Human Cyclophilin B protein (<a href='/en-us/products/unavailable/recombinant-human-cyclophilin-b-protein-ab88801'>ab88801</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 24 kDa

true

Exposure time: 30s

• WB

Project

Western blot - Anti-Cyclophilin B antibody - Loading Control (AB16045)

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 24 kDa

Observed band size: 25 kDa

false

Exposure time: 30s