Anti-IL3RA/CD123 antibody [EPR24481-20]

• Flow Cyt

Lab

Flow Cytometry - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab280355 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left). PBMCs were incubated for 30 mins on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab280355 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (1x 10⁶ in 100 µl at 1.0 μg/ml (1/2000 dilution)) for 30 mins on ice. The cells were simultaneously stained with CD303.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 dilution for 30 mins on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• Flow Cyt

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Flow Cytometry - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

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 Cyt

Supplier Data

Flow Cytometry - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

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.

• IP

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Immunoprecipitation - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

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

All lanes:

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

Predicted band size: 43 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

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

All lanes:

Western blot - Anti-IL3RA/CD123 antibody [EPR24481-20] (ab280355) at 1/1000 dilution

All lanes:

KG-1a (human bone marrow myeloblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-IL3RA/CD123 antibody [EPR24481-20] (AB280355)

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

All lanes:

Western blot - 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:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 43 kDa

Observed band size: 60 kDa

false