IHC image of ERG staining in a section of formalin fixed, paraffin embedded human prostate cancer. The section was pre-treated using pressure cooker heat mediated antigen retrieval with Tris-EDTA (pH9). The section was incubated with unpurified ab214341, 0.16 µg/ml, for 30 mins at room temperature followed by an incubation with a ready to use anti mouse HRP polymer system for 15 mins at room temperature. The section was counterstained with haematoxylin and mounted with permanent mounting media. As a negative control (inset) the same assay was performed on a section of formalin fixed, paraffin embedded, normal human prostate.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

All lanes : Anti-ERG antibody [EPR3864] - Chimeric (ab214341) at 1/1000 dilution (purified)

Lane 1 : Rat heart lysates
Lane 2 : Rat brain lysates
Lane 3 : Mouse brain lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/2000 dilution

Predicted band size: 55 kDa
Observed band size: 55 kDa

Blocking and diluting buffer: 5% NFDM/TBST

Anti-ERG antibody [EPR3864] - Chimeric (ab214341) at 1/5000 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary
Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/2000 dilution

Predicted band size: 55 kDa
Observed band size: 55 kDa

Blocking and diluting buffer: 5% NFDM/TBST 

Lane 1 : Anti-ERG antibody [EPR3864] - Chimeric (ab214341) at 1 µg/ml (unpurified)
Lane 2 : Anti-ERG antibody [EPR3864] (ab92513) at 1 µg/ml (unpurified)

All lanes : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution
Lane 2 : Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 55 kDa


Exposure time: 4 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab214341 (lane 1) and ab92513 (lane 2) overnight at 4°C. Antibody binding was detected using an anti-mouse (lane 1) and anti-rabbit (lane 2) antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human prostatic hyperplasia tissue sections labeling ERG with purified ab214341 at 1:1000 dilution (1.39 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.