Anti-Peroxiredoxin 2/PRP antibody [EPR5155] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Peroxiredoxin 2/PRP antibody [EPR5155] - BSA and Azide free (AB248516)

This data was developed using ab133481, the same antibody clone in a different buffer formulation.

Immunofluorescence analysis of HeLa cells labelling Peroxiredoxin 2/PRP with ab133481 at 1/100 dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Peroxiredoxin 2/PRP antibody [EPR5155] - BSA and Azide free (AB248516)

This data was developed using ab133481, the same antibody clone in a different buffer formulation.Overlay histogram showing HeLa cells stained with ab133481 (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 (ab133481, 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

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Peroxiredoxin 2/PRP antibody [EPR5155] - BSA and Azide free (AB248516)

Immunofluorescent analysis of 100% methanol-fixed-fixed 0.1% Triton X-100 permeabilized Hap1 WT and Hap1-PRDX2 KO cells labelling Peroxiredoxin 2 with ab133481 at 0.2 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 [EPR5155] - BSA and Azide free (AB248516)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133481).

Flow cytometry overlay histogram showing wild-type HAP1 (green line) and PRDX2 knockout HAP1 stained with ab133481 (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 (ab133481) (1x 10⁶ in 100μl at 0.008 μg/ml (1/14375 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.

• WB

Lab

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5155] - BSA and Azide free (AB248516)

This data was developed using the same antibody clone in a different buffer formulation (ab133481).

Lanes 1- 2 : Merged signal (red and green). Green - ab133481 observed at 22 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab133481 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. ab133481 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 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 [EPR5155] (<a href='/en-us/products/primary-antibodies/peroxiredoxin-2-prp-antibody-epr5155-ab133481'>ab133481</a>) at 1/5000 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

false

• WB

Unknown

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5155] - BSA and Azide free (AB248516)

This data was developed using ab133481, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5155] (<a href='/en-us/products/primary-antibodies/peroxiredoxin-2-prp-antibody-epr5155-ab133481'>ab133481</a>) at 1/50000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

HepG2 cell lysate at 10 µg

Lane 3:

LnCaP cell lysate at 10 µg

Lane 4:

U937 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 22 kDa

false

• WB

Lab

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5155] - BSA and Azide free (AB248516)

This data was developed using ab133481, the same antibody clone in a different buffer formulation.

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 - ab133481 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133481 was shown to specifically react with Peroxiredoxin 2/PRP when Peroxiredoxin 2/PRP knockout samples were used. Wild-type and Peroxiredoxin 2/PRP knockout samples were subjected to SDS-PAGE. ab133481 and ab8245 (loading control to GAPDH) were diluted 1/50 000 and 1/10 000 respectively 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Peroxiredoxin 2/PRP antibody [EPR5155] (<a href='/en-us/products/primary-antibodies/peroxiredoxin-2-prp-antibody-epr5155-ab133481'>ab133481</a>)

Predicted band size: 22 kDa

false