Recombinant Anti-PRAME antibody [EPR20330] (ab219650)

Immunohistochemical analysis of paraffin-embedded human melanoma tissue labeling PRAME with ab219650 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on human melanoma (PMID: 9047241).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab219650 staining PRAME 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.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed MeWo (Human malignant melanoma cell line) cells labeling PRAME with ab219650 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled 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. 

 

 

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

Lane 1 : Human ovary cancer lysate
Lane 2 : A-375 (Human malignant melanoma cell line) whole cell lysate
Lane 3 : Human testis lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 57 kDa
Observed band size: 57 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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.

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.

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.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling PRAME with ab219650 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on human testis (PMID: 9047241).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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.

Anti-PRAME antibody [EPR20330] (ab219650) at 1/1000 dilution + MeWo (Human malignant melanoma cell line) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/4000 dilution

Predicted band size: 57 kDa
Observed band size: 57 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling PRAME with ab219650 at 1/16000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Negative control: No staining on human pancreas (PMID: 9047241).

Counter stained with Hematoxylin.

 

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.