Anti-PRAME antibody [EPR20330](AB219650)

PRAME immunofluorescence with PRAME antibody ab219650 in K562 cells, with negative expression in Ramos cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab219650 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

This product also work with 100% methanol (5 min) fixation under the same testing conditions.

PRAME was immunoprecipitated from 0.35 mg of MeWo (Human malignant melanoma cell line) whole cell lysate with ab219650 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab219650 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: MeWo whole cell lysate 10 μg (Input).

Lane 2: ab219650 IP in MeWo whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab219650 in MeWo whole cell lysate.

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

Exposure time: 1 second.

All lanes: Immunoprecipitation - Anti-PRAME antibody [EPR20330] (ab219650)

Predicted band size: 57 kDa

PRAME western blot with PRAME antibody ab219650.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2: 5 seconds; Lane 3: 1 minute.

All lanes: Western blot - Anti-PRAME antibody [EPR20330] (ab219650) at 1/1000 dilution

Lane 1: Human ovary cancer lysate at 20 µg

Lane 2: A-375 (Human malignant melanoma cell line) whole cell lysate at 20 µg

Lane 3: Human testis lysate at 20 µg

Secondary

All lanes: Western blot - VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 57 kDa

Observed band size: 57 kDa

PRAME western blot with PRAME antibody ab219650.

Different batches of ab219650 were tested on MeWo (Human malignant melanoma fibroblast) whole cell lysate at 0.1 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 50 kDa.

All lanes: Western blot - Anti-PRAME antibody [EPR20330] (ab219650)

Predicted band size: 57 kDa

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MeWo (Human malignant melanoma cell line) cells labeling PRAME with ab219650 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing mostly nuclear staining on MeWo cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A-375 (Human malignant melanoma cell line) cells labeling PRAME with ab219650 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing mostly nuclear staining on A-375 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.