All lanes : Anti-Lin28A antibody [EPR4640] (ab124765) at 1/10000 dilution (Purified)

Lane 1 : NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysates
Lane 2 : JAR (Human placenta choriocarcinoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 23 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human liver tissue sections labeling Lin28A with purified ab124765 at 1/50 dilution (12 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-Lin28A antibody [EPR4640] (ab124765) at 1/10000 dilution (unpurified)

Lane 1 : NCCIT cell lysate
Lane 2 : JAR cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 23 kDa

Immunocytochemistry/ Immunofluorescence analysis of JAR (Human placenta choriocarcinoma epithelial cell) cells labeling Lin28A with purified ab124765 at 1/500 dilution (1.2 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of NCCIT (Human pluripotent embryonic carcinoma epithelial cell) cells labeling Lin28A with purified ab124765 at 1/60 dilution (10 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).

ab124765 (purified) at 1/30 dilution (2ug) immunoprecipitating Lin28A in NCCIT whole cell lysate. NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab124765 & NCCIT whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab124765 in NCCIT whole cell lysate
For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

ab124765 (unpurified), at 1/100 dilution, staining Lin28A in paraffin-embedded Human seminoma tissue by Immunohistochemistry.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

ab124765 (unpurified), at 1/100 dilution, staining Lin28A in NCCIT cells by Immunofluorescence.

ab124765 (unpurified) showing positive staining in Glioma tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

ab124765 (unpurified) showing positive staining in Dysgerminoma tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

ab124765 (unpurified) showing positive staining in Embryonal carcinoma tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

ab124765 (unpurified) showing negative staining in Normal brain tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.