Anti-CRTAM antibody [EPR23786-12]

• Flow Cyt

Unknown

Flow Cytometry - Anti-CRTAM antibody [EPR23786-12] (AB272723)

Flow cytometric analysis of Human peripheral blood mononuclear cells (PBMC) (treated with 1 ng/mL PMA and 0.2 μM Ionomycin for 5h) labelling CRTAM with ab272723 at 1/500 dilution (0.1ug) (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left).

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 ab272723. Then stained with anti-CD8 alpha conjugated to Pacific blue.

Gated on viable cells.

• Flow Cyt

Lab

Flow Cytometry - Anti-CRTAM antibody [EPR23786-12] (AB272723)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) (top) or PBMCs treated with 1ng/ml PMA 18 hr (bottom), with ab272723 (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 ab272723 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (1x 10⁶ in 100 µl at 1.0 μg/ml (1/2110 dilution)) for 30 mins on ice. The cells were simultaneously stained with CD8.

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.

• WB

Lab

Western blot - Anti-CRTAM antibody [EPR23786-12] (AB272723)

The molecular weight observed is consistent with what has been described in the literature (PMID : 10811014, 23637756, 16300832).

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

All lanes:

Western blot - Anti-CRTAM antibody [EPR23786-12] (ab272723) at 1/1000 dilution

Lane 1:

Untreated human peripheral blood mononuclear cell (PBMC) whole cell lysate at 20 µg

Lane 2:

Human PBMC treated with 1ng/mL phorbol-12-myristate-13-acetate (PMA) (<a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>) and 0.2 μM ionomycin (<a href='/en-us/products/biochemicals/ionomycin-ca2-salt-ca2-ionophore-ab120116'>ab120116</a>) for 5 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 45 kDa

Observed band size: 70 kDa,85 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-CRTAM antibody [EPR23786-12] (AB272723)

The molecular weight observed is consistent with what has been described in the literature (PMID : 10811014, 23637756, 16300832).

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

All lanes:

Western blot - Anti-CRTAM antibody [EPR23786-12] (ab272723) at 1/1000 dilution

Lane 1:

Mouse thymus tissue lysate at 40 µg

Lane 2:

Rat cerebellum tissue lysate at 40 µg

Secondary

All lanes:

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

Predicted band size: 45 kDa

Observed band size: 70 kDa

false

Exposure time: 3min