Anti-Pea3 antibody [1A2G3] (ab70425)

ab70425 staining Pea3 in Human bladder transitional cell carcinoma tissue by Immunohistochemistry (Formalin/ PFA-fixed paraffin-embedded tissue sections). The sections were fixed with formalin overnight and subjected to heat mediated antigen retrieval in citrate buffer prior to blocking with 5% BSA for 30 minutes. The primary antibody was diluted 1/200 (5% BSA in PSA) and incubated with the sample for 1 hour at 20°C. An HRP-conjugated Goat anti-Mouse polyclonal was used undiluted as the secondary antibody.

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All lanes : Anti-Pea3 antibody [1A2G3] (ab70425) at 1/500 dilution

Lane 1 : Truncated Trx-Pea3 recombinant protein
Lane 2 : Full length Pea3 hIgGFc transfected CHO-K1 cell lysate

Predicted band size: 54 kDa
Observed band size: 24,60 kDa why is the actual band size different from the predicted?

Anti-Pea3 antibody [1A2G3] (ab70425) at 1/2000 dilution + Cell lysates prepared from K562 cells at 100 µg

Secondary
HRP-conjugated Goat polyclonal to mouse IgG1


Predicted band size: 54 kDa

Overlay histogram showing K562 cells stained with ab70425 (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 (ab70425, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in K562 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.