Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade

• IHC

Lab

Immunohistochemistry - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

Immunohistochemical analysis of formalin fixed paraffin embedded human testis labelling androgen receptor with ab108341 at a concentration of 0.19 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5 for 32 mins.

ab108341 Anti-androgen receptor antibody [ER179(2)] was incubated for 16 mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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.

• ChIP

Lab

ChIP - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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.

• WB

Lab

Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/5000 dilution

All lanes:

LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 99 kDa

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• WB

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Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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.

All lanes:

Western blot - 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 at 15 µg

Lane 2:

LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method at 15 µg

Lane 3:

22Rv1 (Human prostate carcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method at 15 µg

Lane 4:

22Rv1 (Human prostate carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 99 kDa

Observed band size: 120 kDa

false

Exposure time: 20s

• WB

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Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

All lanes:

Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/1000 dilution

Lane 1:

T47 D cell lysate at 10 µg

Lane 2:

LnCaP cell lysate at 10 µg

Predicted band size: 99 kDa

false

• WB

Lab

Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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.

All lanes:

Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/1000 dilution

Lane 1:

LnCAP whole cell lysate (positive control) at 20 µg

Lane 2:

PC3 whole cell lysate (negative control) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 99 kDa

Observed band size: 120 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 LNCaP (Human prostate carcinoma epithelial cell) cells cultured in phenol red free medium and 5% charcoal stripped FBS for 3 days then treated with DHT (10 nM 4h), and 5 µg of ab108341 [ER179(2)]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Unknown

Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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

Observed MW; 110 kDa

All lanes:

Western blot - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (ab108341) at 1/1000 dilution

Lane 1:

Mouse testis lysates at 20 µg

Lane 2:

Rat testis lysates at 20 µg

Lane 3:

Mouse liver lysates at 20 µg

Lane 4:

Rat liver lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 99 kDa

false

Exposure time: 20s

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Androgen Receptor antibody [ER179(2)] - ChIP Grade (AB108341)

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