All lanes : Anti-RNA Helicase A antibody [EPR13521] (ab183731) at 1/10000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 3 : THP-1 (Human monocytic leukemia monocyte) 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: 141 kDa
Observed band size: 128,141 kDa why is the actual band size different from the predicted?

Blocking/Diluting Buffer and concentration: 5% NFDM/TBST

The observed bands are consistent with what are described in PMID: 28337295

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling RNA Helicase A with purified ab183731 at 1/20 dilution (5 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).

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling RNA Helicase A with purified ab183731 at 1/50 dilution (2.2 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) 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney carcinoma tissue sections labeling RNA Helicase A with purified ab183731 at 1/400 dilution (0.28 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.

Flow cytometry analysis of HeLa cells using ab183731 (unpurified) at a 1/10 dilution (red) or a Rabbit monoclonal IgG (negative) (green). Goat anti rabbit IgG (FITC) secondary used at a 1/150 dilution.

Immunocytochemistry analysis of HeLa cells (fixative -20℃ Acetone) labeling RNA Helicase A with ab183731 (unpurified) at a 1/250 dilution. Goat anti rabbit IgG (Alexa Fluor^® 488) secondary used at a 1/200 diution.

Immunohistochemical analysis of paraffin embedded Human lung adenocarcinoma tissue labeling RNA Helicase A with ab183731 (unpurified) at a 1/100 dilution. Prediluted HRP conjugated Rabbit IgG secondary used. Counterstained with Hematoxylin. Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.