Anti-ERG antibody [EPR3864] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

Flow cytometry overlay histogram showing left THP-1 positive cells and right negative HCT116 stained with ab92513 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab92513) (1x 106 in 100μl at 0.04μg/ml (1/54000)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

Immunohistochemical staining of paraffin embedded human kidney with purified ab92513 at a working dilution of 1/1000. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

Intracellular Flow Cytometry analysis of THP-1 (human monocytic leukemia cell line) cells labeling ERG with purified ab92513 at 1/1000 dilution (1ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) (ab172730) was used as the isotype control, Cell without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

ab92513 staining ERG in THP-1 cells, with negative expression in HCT116 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 ab92513 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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

ERG and GSTP1 immunostainings of human prostate cancer samples using ab92513.

Representative immunohistochemical images of prostate cancer samples are shown that were positive for ERG and negative for GSTP1 (A), positive for both ERG and GSTP1 (B), negative for both ERG and GSTP1(C), and negative for ERG and positive for GSTP1 (D). The internal staining control for ERG is the endothelium (arrows) and for GSTP1 the stromal and/or basal cells of normal prostate glands. N, normal prostate gland; S, Stroma; T, tumor gland. Scale bars equal 100μm

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Image from Litovkin K et al. PLoS One. 2015;10(6):e0130651. Fig 5.; doi: 10.1371/journal.pone.0130651.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

Immunohistochemical analysis of formalin-fixed paraffin-embedded human kidney labelling ERG with ab92513 at a concentration of 1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab92513 anti ERG antibody was incubated at 37°C for 16min. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

Immunohistochemical analysis of paraffin embedded Human Prostatic adenocarcinoma stage 3 tissue using unpurified ab92513 showing +ve staining.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

Immunofluorescence staining of THP-1 (human monocytic leukemia cell line) cells with purified ab92513 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab92513 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

Functional characterization and detection of genetic alterations in GEDI-captured cells. The TMPRSS2 : ERG fusion protein is detected in GEDI-captured circulating tumor cells (CTCs) from a castrate-resistant prostate cancer (CRPC) patient. PSMA-captured CTCs were stained on the device with ab92513. Representative examples of PSMA+/CD45− CTCs are shown, two of which are positive for ERG. Scale bars : 10 microns.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513).

Image from Kirby BJ et al. PLoS One. 2012;8(12):e83903. Fig 4.; doi: 10.1371/journal.pone.0035976.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92513). Tissue Microarrays stained for Anti-ERG antibody [EPR3864] using ab92513 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The section was incubated with ab92513 for 30 mins at room temperature followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-ERG antibody [EPR3864] - BSA and Azide free (AB214796)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.