Anti-eIF5A antibody [EP526Y] (ab32443) at 1/10000 dilution + Jurkat cell lysate.

Predicted band size: 18 kDa
Observed band size: 18 kDa

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling eIF5A with purified ab32443 at 1/500. Cells were fixed with 4% Paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue). 

Secondary Only Control: PBS was used instead of the primary antibody as the negative control

ab32443 at a 1:250 dilution staining eIF5A in human adenocarcinoma of uterus using Immunohistochemistry, Paraffin Embedded Tissue.

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

All lanes : Anti-eIF5A antibody [EP526Y] (ab32443) at 1/2000 dilution

Lane 1 : Recombinant Human eIF5A protein (ab87457)
Lane 2 : Recombinant Human eIF5A2 protein (ab99140)

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Developed using the ECL technique.

Predicted band size: 18 kDa
Observed band size: 15,20 kDa

why is the actual band size different from the predicted?


Exposure time: 20 seconds

Blocking buffer: 5% NFDM/TBST

This antibody shows low affinity in recognizing eIF5A2 recombinant protein.

Overlay histogram showing Jurkat cells stained with ab32443 (red line). The cells were fixed with 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 (ab32443, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (0.5µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.