All lanes : Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/1000 dilution

Lane 1 : Mouse testis lysates
Lane 2 : Rat testis lysates
Lane 3 : Mouse liver lysates
Lane 4 : Rat liver lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 99 kDa


Exposure time: 20 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Observed MW; 110 kDa

Immunohistochemistry (paraffin-embedded sections) analysis of mouse testis tissue labelling Androgen with ab108341 at 0.12 µg/mL for 30mins at room temperature. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 epitope retrieval solution 2) for 20 mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody (1/4000). Counterstained with hematoxylin. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

All lanes : Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/5000 dilution

Lane 1 : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method
Lane 2 : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method
Lane 3 : 22Rv1 (Human prostate carcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method
Lane 4 : 22Rv1 (Human prostate carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 99 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?


Exposure time: 20 seconds

Blocking/Diluting buffer: 5% NFDM/TBST

The androgen receptor variant band detected in 22RV1 cells is reported by PMID: 22315407.

We recommend you to try both RIPA and 1% SDS Hot lysis preparation methods to get desired bands.

Immunocytochemistry/ Immunofluorescence analysis of LNCaP (Human prostate carcinoma epithelial cell) cells labeling Androgen receptor with Purified ab108341 at 1:500 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor ® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor ® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Chromatin was prepared from LNCaP cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab108341 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are commercial primers from Paper (PMID: 25802280)
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue sections labeling Androgen receptor with Purified ab108341 at 1:100 dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). 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.

Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/5000 dilution (purified) + LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 99 kDa

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/1000 dilution (unpurified)

Lane 1 : LnCAP whole cell lysate (positive control)
Lane 2 : PC3 whole cell lysate (negative control)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 99 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab108341 observed at 120 kDa. Red - loading control, ab181602, observed at 37 kDa.

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 Licor blocking buffer before being incubated with ab108341 and ab181602 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at a 1:10000 dilution for 1hr at room temperature and then imaged.

Unpurified ab108341, at 1/100, staining Androgen Receptor in LnCaP cells by Immunofluorescence.

All lanes : Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/1000 dilution (unpurified)

Lane 1 : T47 D cell lysate
Lane 2 : LnCaP cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 99 kDa

Unpurified ab108341, at 1/250, staining Androgen Receptor in paraffin-embedded Human prostatic adenocarcinoma tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab108341, at 1/250, staining Androgen Receptor in paraffin-embedded Human prostate tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab108341 showing positive staining in Normal testis tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab108341 showing positive staining in Prostatic carcinoma T3 tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified  ab108341 showing positive staining in Breast carcinoma tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab108341 showing negative staining in Normal liver tissue. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.