Anti-Peroxiredoxin 1/PAG antibody

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Peroxiredoxin 1/PAG antibody (AB15571)

IHC image of ab15571 staining in human liver carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab15571, 1/100 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Peroxiredoxin 1/PAG antibody (AB15571)

ICC/IF image of ab15571 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab15571, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Peroxiredoxin 1/PAG antibody (AB15571)

Immunocytochemistry/ Immunofluorescence analysis of Peroxiredoxin 1/PAG was performed using 70% confluent log phase T-47D cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ab15571 at 1/250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor^® 488 conjugate at a dilution of 1/2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Rhodamine Phalloidin at 1/300. Panel d represents the merged image showing cytosolic and nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

• WB

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Western blot - Anti-Peroxiredoxin 1/PAG antibody (AB15571)

Western blot analysis of Peroxiredoxin 1//PAG was performed by loading 25ug of various whole cell lysates onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were probed with ab15571 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody for at least one hour. Membranes were washed and chemiluminescent detection performed.

All lanes:

Western blot - Anti-Peroxiredoxin 1/PAG antibody (ab15571) at 1/1000 dilution

All lanes:

Cell line indicated at 25 µg

Secondary

All lanes:

HRP-conjugated Goat anti-rabbit at 1/20000 dilution

Predicted band size: 22 kDa

Observed band size: 20 kDa,25 kDa

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• WB

Lab

Western blot - Anti-Peroxiredoxin 1/PAG antibody (AB15571)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : Peroxiredoxin 1/PAG knockout HAP1 cell lysate (20 μg)
Lane 3 : A431 cell lysate (20 μg)
Lane 4 : Jurkat cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab15571 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab15571 was shown to recognize Peroxiredoxin 1/PAG when Peroxiredoxin 1/PAG knockout samples were used, along with additional cross-reactive bands. Wild-type and Peroxiredoxin 1/PAG knockout samples were subjected to SDS-PAGE. ab15571 and ab8245 (loading control to GAPDH) were diluted to 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) andGoat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Peroxiredoxin 1/PAG antibody (ab15571)

Predicted band size: 22 kDa

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