Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (AB128953)

ab128953 at a 1/250 dilution staining Peroxiredoxin 3/PRDX3 in paraffin embedded Human liver tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (AB128953)

Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Peroxiredoxin 3/PRDX3 with purified ab128953 at 1/1000 dilution (1 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (AB128953)

ab128953 at a 1/250 dilution staining Peroxiredoxin 3/PRDX3 in paraffin embedded Human endometrium adenocarcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (AB128953)

Peroxiredoxin 3/PRDX3 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg with ab128953 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : ab128953 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab128953 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Upper bands could be ubiquitinated forms (PMID : 21795677)

All lanes:

Immunoprecipitation - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (ab128953)

Predicted band size: 28 kDa

Observed band size: 26 kDa

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• WB

Lab

Western blot - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (AB128953)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : Peroxiredoxin 3/PRDX3 knockout HAP1 cell lysate (20 μg)
Lane 3 : MCF7 cell lysate (20 μg)
Lane 4 : A431 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab128953 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab128953 was shown to specifically react with Peroxiredoxin 3/PRDX3 in wild-type HAP1 cells. No band was observed when Peroxiredoxin 3/PRDX3 knockout samples were examined. Wild-type and Peroxiredoxin 3/PRDX3 knockout samples were subjected to SDS-PAGE. ab128953 and ab8245 (loading control to GAPDH) were both diluted 1/10,000 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 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (ab128953)

Predicted band size: 28 kDa

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• WB

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Western blot - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (AB128953)

All lanes:

Western blot - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (ab128953) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T (Human embryonic kidney epithelial cell) cell lysate at 10 µg

Lane 3:

MCF7 cell lysate at 10 µg

Lane 4:

A431 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 28 kDa

Observed band size: 26 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-Peroxiredoxin 3/PRDX3 antibody [EPR8115] (AB128953)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.