Anti-Peroxiredoxin 2/PRP antibody [EPR5154]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Immunofluorescent analysis of 100% methanol-fixed 0.1% Triton X-100 permeabilized Hap1 WT and Hap1-PRDX2 KO cells labelling Peroxiredoxin 2 with ab109367 at at 5 μg/ml concentration followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 μg/ml dilution (Green). Image showing cytoplasmic staining in Hap1 WT cell line. ab7291 Anti-alpha Tubulin antibody [DM1A] was used to counterstain tubulin at 1/1000 dilution (Magenta). The nuclear counterstain was DAPI (Blue).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Flow cytometry overlay histogram showing wild-type HAP1 (green line) and PRDX2 knockout HAP1 stained with ab109367 (magenta line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab109367) (1x 10⁶ in 100μl at 0.04 μg/ml (1/21200 dilution)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 dilution for 30min at 22°C.

Isotype control antibody was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control in HAP1 WT cells (black line) and HAP1-PRDX2 KO cells (grey line), used at the same concentration and conditions as the primary antibody.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Overlay histogram showing HeLa cells fixed in 80% methanol and stained with purified ab109367 at a dilution of 1/200 (red line). The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit at a dilution of 1/500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Overlay histogram showing HeLa cells stained with unpurifiedab109367 (red line). The cells were fixed with 80% methanol (5 min) 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 (ab109367, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Immunofluorescent staining of Peroxiredoxin 2/PRP in HeLa cells using unpurified ab109367 at 1/250 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Immunohistochemical analysis of Peroxiredoxin 2/PRP in paraffin-embedded Human stomach tissue using unpurified ab109367 at 1/1000 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Immunohistochemical analysis of Peroxiredoxin 2/PRP in paraffin-embedded Human prostatic hyperplasia tissue using unpurified ab109367 at 1/1000 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Immunohistochemical staining of paraffin embedded human endometrial carcinoma with purified ab109367 at a working dilution of 1/500. The secondary antibody used is ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Immunofluorescence staining of HeLa cells with purified ab109367 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 100% methanol and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab109367 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

• ICC/IF

AbReview31000****

Immunocytochemistry/ Immunofluorescence - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Unpurified ab109367 (1/500) staining Peroxiredoxin 2/PRP in asynchronous HeLa cells (green). Cells were fixed in methanol and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

• WB

Lab

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (ab109367) at 1/10000 dilution

Lane 1:

HEK293 whole cell lysate at 10 µg

Lane 2:

LNCaP whole cell lysate at 10 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-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

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• WB

Lab

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Lanes 1- 2 : Merged signal (red and green). Green - ab109367 observed at 22 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109367 was shown to react with Peroxiredoxin 2/PRP in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266392 (knockout cell lysate ab257041) was used. Wild-type HEK-293T and PRDX2 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109367 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (ab109367) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

PRDX2 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human PRDX2 (Peroxiredoxin 2/PRP) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-prdx2-peroxiredoxin-2-prp-knockout-hek-293t-cell-line-ab266392'>ab266392</a>)

Predicted band size: 22 kDa

Observed band size: 22 kDa

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• WB

Lab

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : Peroxiredoxin 2/PRP knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : LnCaP cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109367 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109367 was shown to specifically react with Peroxiredoxin 2/PRP when Peroxiredoxin 2/PRP knockout samples were used. Wild-type and Peroxiredoxin 2 knockout samples were subjected to SDS-PAGE. ab109367 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (ab109367)

Predicted band size: 22 kDa

false

• WB

Lab

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (ab109367) at 1/10000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Rat brain lysate at 10 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-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

• WB

Unknown

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (AB109367)

All lanes:

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (ab109367) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

LnCaP cell lysate at 10 µg

Lane 4:

SH-SYSY cell lysate at 10 µg

Secondary

All lanes:

HRP labelled Goat anti-Rabbit at 1/2000 dilution

Predicted band size: 22 kDa

false