Anti-eIF5A2 + eIF5A antibody [EPR7411-6]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-eIF5A2 + eIF5A antibody [EPR7411-6] (AB126735)

Overlay histogram showing HeLa cells stained with ab126735 (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 (ab126735, 1/100 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) (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF5A2 + eIF5A antibody [EPR7411-6] (AB126735)

ab126735 at 1/100 dilution, labeling eIF5A2 + eIF5A in Formalin fixed Paraffin-embedded Human ovarian carcinoma tissue by Immunohistochemistry.

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

• WB

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Western blot - Anti-eIF5A2 + eIF5A antibody [EPR7411-6] (AB126735)

All lanes:

Western blot - Anti-eIF5A2 + eIF5A antibody [EPR7411-6] (ab126735) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

A431 cell lysate at 10 µg

Lane 4:

CaCo-2 cell lysate at 10 µg

Lane 5:

HepG2 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated at 1/2000 dilution

Predicted band size: 17 kDa

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• WB

Lab

Western blot - Anti-eIF5A2 + eIF5A antibody [EPR7411-6] (AB126735)

Blocking buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-eIF5A2 + eIF5A antibody [EPR7411-6] (ab126735) at 1/2000 dilution

Lane 1:

Western blot - Recombinant Human eIF5A protein (Tag Free) (<a href='/en-us/products/proteins-peptides/recombinant-human-eif5a-protein-ab87457'>ab87457</a>) at 0.01 µg

Lane 2:

Western blot - Recombinant Human eIF5A2 protein (His tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-eif5a2-protein-ab99140'>ab99140</a>) at 0.01 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: 17 kDa

Observed band size: 15 kDa,20 kDa

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Exposure time: 20s

• OI-RD Scanning

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OI-RD Scanning - Anti-eIF5A2 + eIF5A antibody [EPR7411-6] (AB126735)

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.