Recombinant Anti-IL3RA/CD123 antibody [EPR24481-20] (ab280355)

All lanes : Anti-IL3RA/CD123 antibody [EPR24481-20] (ab280355) at 1/1000 dilution

Lane 1 : Human tonsil tissue lysate at 40 µg
Lane 2 : HDLM-2 (human hodgkin lymphoma ) whole cell lysate at 20 µg
Lane 3 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Secondary
Lane 1 : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution
Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 43 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID:30647406).

Negative control: K-562 cell (PMID: 30647406)

Exposure time: 37 seconds

 

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling IL3RA/CD123 with ab280355 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human tonsil (PMID: 27340692, PMID:15728234, PMID:28205524). The section was incubated with ab280355 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HDLM-2 cells labelling IL3RA/CD123 with ab280355 at 1/1000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in HDLM-2 cells.

Negative control: K-562 (PMID: 26531164).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at a 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

 

Flow cytometric analysis of K-562(Human chronic myelogenous leukemia lymphoblast, Left)/ HDLM-2(Human Hodgkin lymphoma, Right) cells labelling IL3RA/CD123 with ab280355 at 1/500 dilution (0.1ug)/ red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: K-562 cell (PMID: 26531164).

Gated on viable cells.

IL3RA/CD123 was immunoprecipitated from 0.35 mg HDLM-2 (human hodgkin lymphoma ) whole cell lysate 10 µg with ab280355 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280355 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HDLM-2 whole cell lysate 10 µg

Lane 2: ab280355 IP in HDLM-2 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280355 in HDLM-2 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 32 seconds

 

Anti-IL3RA/CD123 antibody [EPR24481-20] (ab280355) at 1/1000 dilution + KG-1a (human bone marrow myeloblast) whole cell lysate at 20 µg

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

Predicted band size: 43 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID:30647406).

Exposure time: 103 seconds

Immunohistochemical analysis of paraffin-embedded human Hodgkin's lymphoma tissue labeling IL3RA/CD123 with ab280355 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human Hodgkin's lymphoma. The section was incubated with ab280355 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Flow cytometric analysis of human peripheral blood mononuclear cell (PBMC) cells labelling IL3RA/CD123 with ab280355 at 1/500 dilution (0.1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with rabbit IgG or ab280355. Then stained with anti-HLA-DR conjugated to PE.

Gated on viable cells.