Androgen Receptor antibody

Catalog No. ABIN1871059

Product Details anti-Androgen Receptor Antibody

Target: Androgen Receptor (AR)

Reactivity: Human, Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This Androgen Receptor antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunocytochemistry (ICC)

Characteristics: Expected molecular weight of target: 919-920 kDa

Purification: Affinity purification

Immunogen: A synthetic peptide of human AR

Isotype: IgG

Application Details

Application Notes: WB 1:500-1:2000, IHC 1:50-1:200, ICC 1:50-1:200, IF 1:50-1:200

Restrictions: For Research Use only

Independent Validation (IF)

Validation #101745 (Immunofluorescence)

by: School of Science and Engineering, Tulane University

No.: #101745

Date: 12/21/2017

Antigen: Androgen Receptor

Lot Number: 09Q266222

Method validated: Immunofluorescence

Positive Control: prostate from inducible ARlacZ mouse

Negative Control: secondary antibody only

Notes:

Passed. Despite some weak non-specific staining, this antibody robustly labels the AR at a dilution of 1:100.

Validation Images

IF staining of prostate from inducible AR-lacZ mouse with ABIN1871059 shows robust staining for the AR at 1:50 dilution (A) and colocalization with nuclear DAPI staining (B, C). Staining is robust also at 1:100 (D), 1:200 (E), and 1:250 (F) dilutions. Non-specific signal in the secondary antibody-only control is both extracellular and nuclear. Exposure 5x of that used to image ABIN1871059 was used in G to capture the non-specific signal in the TRITC channel. H shows DAPI imaged at 100ms and I show that the non-specific signal is both extracellular and nuclear, as shown by the partial colocalization of the non-specific signal with DAPI.

Full Methods

Primary Antibody: ABIN1871059

Secondary Antibody: goat anti-rabbit Cy3 conjugated antibody (Jackson ImmunoResearch)

Full Protocol:

• Prostate Tissue from ARlacz transgenic mouse or wild type mouse is fixed via transcardial perfusion with 10% formalin and postfixed at 4°C overnight. Tissue from the transgenic mouse is used for a positive control while tissue from the wild-type mouse is used with the AR antibody.
• Dewaxing and rehydration:
• CitriSolv (Decon Laboratories, 1601H, 11068) 2x for 5min.
  • 100% ethanol 2x for 3min.
  • 95% ethanol 2x for 3min.
  • 70% ethanol 2x for 3min.
  • Rinse well in distilled H₂O.
• HIER:
• Dilute 10x citrate buffer pH6.0 1:10 with dH₂O and place in cases.
  • Place one slide into each case in the preheated pressure cooker (Hamilton Beach).
  • Antigen unmasking for 40min at 92-99°C.
  • Remove slides from pressure cooker (but leave them in the cases with citrate buffer). Let slides cool down for 20min to approximately 30°C.
• Wash sections 2x for 5min with PBST rocking in a coplin jar.
• Wash sections for 5min with PBS rocking in a coplin jar.
• Permeabilize sections 10min in PBS + 0.2 Triton X-100 rocking in a coplin jar.
• Wash sections 3x for 5min with PBST rocking in a coplin jar.
• Aspirate liquid, circle tissue with a PAP pen and let dry.
• Block each section with 200µl blocking solution (PBS containing 1% BSA, 5% normal goat serum, and 0.02% Triton X-100) for 1h at RT.
• Aspirate blocking solution.
• Incubate sections with 100µl primary
• rabbit anti-androgen receptor antibody (antibodies-online, ABIN1871059, 09Q266222) diluted 1:50, 1:100, 1:200, and 1:250 in 1:10 blocking solution ON at 4°C.
  • rabbit anti-β-galactosidase antibody (Life Technologies, A1132, lot 1720092) diluted 1:500 in 1:10 blocking solution (in PBS) ON at 4°C.
  • Include a no primary antibody negative controls.
• Aspirate the liquid.
• Wash sections 2x for 5min with PBST.
• Wash sections for 5min with PBST.
• Incubate sections in the dark with secondary goat anti-rabbit Cy3 conjugated antibody (Jackson ImmunoResearch) diluted 1:400 in PBS for 1h at RT.
• Aspirate the liquid in the dark.
• Wash sections in the dark 2x for 5min with PBST.
• Wash sections in the dark for 5min with PBST.
• Nuclear counterstaining with 14.3mM DAPI diluted 1:50000 in PBS for 5min at RT in the dark.
• Wash sections in the dark 2x for 5min with PBST.
• Mount sections in mounting medium (Invitrogen, P36930, lot 1811419) put 1 drop of medium on each section and cover with a coverslip in the dark; seal your slides with nail polish to avoid dehydration. Store sections in the dark at 4°C.
• Visualization on a microscope (Nikon, Eclipse Ti) with 20x objective; exposure time TRITC AR 100ms, DAPI 100ms.

Experimental Notes:

• ABIN1871059 shows robust staining for the AR at all dilutions tried (A, D, E, and F). Dilution series was determined by manufacturer's recommendations.

• The staining for the AR is clearly nuclear. This is shown by colocalization with the nuclear stain DAPI (B and C). Additionally, the staining pattern seen with the AR antibody is similar to the staining pattern of β-galactosidase seen in the ARlacz transgenic mouse. The ARlacz transgenic mouse expresses β-galactosidase (β-gal) under the control of the AR promoter. Therefore, β-gal is expressed wherever the endogenous AR is expressed.

• Exclusion of the primary antibody eliminates nuclear staining, demonstrating that the AR staining is specific. Panels G and H show a prostate that was exposed to secondary antibody, but not primary antibody. The exposure time in Panel G is the same as that for the images shown in the previous figures. The exposure time in Panel H is 5x that shown in the previous figures. Although this isn’t the best negative control to use, we do not have prostate or global AR knockout mice to use for a control.

Handling

Format: Liquid

Concentration: 1 mg/mL

Buffer: PBS with 0.02 % sodium azide, 50 % glycerol, pH 7.4

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Avoid repeated freeze/thaw cycles.

Storage: -20 °C

Storage Comment: Store at -20°C.

Target Details for Androgen Receptor

Target: Androgen Receptor (AR)

Alternative Name: AR (AR Products)

Synonyms: AIS antibody, DHTR antibody, HUMARA antibody, HYSP1 antibody, KD antibody, NR3C4 antibody, SBMA antibody, SMAX1 antibody, TFM antibody, AW320017 antibody, Tfm antibody, Andr antibody, AR antibody, androgen receptor antibody, AR antibody, Ar antibody, ar antibody

Molecular Weight: 110 kDa

Gene ID: 367

UniProt: P10275

Pathways: Nuclear Receptor Transcription Pathway, Intracellular Steroid Hormone Receptor Signaling Pathway, Steroid Hormone Mediated Signaling Pathway, Regulation of Intracellular Steroid Hormone Receptor Signaling, Nuclear Hormone Receptor Binding, Chromatin Binding