Image 1: Structure of the Androgen Receptor target protein.

Androgen Receptor Target Introduction

Protein Function

• Androgen Receptor (AR) belongs to the nuclear receptor superfamily of steroid receptors and acts as a transcription factor to regulate gene expression in eukaryotes, playing crucial roles in reproduction, skeletal muscle, cardiovascular, nervous, immune, hematopoietic system development, and homeostasis.
• AR contains four functional domains: N-terminal transcriptional regulatory domain (NTD), highly conserved DNA binding domain (DBD), hinge domain, and ligand binding domain (LBD). AR has many signal sequences, including transcription activation signals AF1, AF2, nuclear localization signal (NLS), and nuclear export signal (NES).
• In the absence of ligand, AR is in the cytoplasm, bound to heat shock proteins (such as HSP90, HSP70) and other molecular chaperone proteins. Upon binding of androgens to AR, conformational changes occur, leading to dissociation of AR from molecular chaperones and exposure of NLS. Subsequently, the androgen/AR complex is transported to the nucleus, undergoes dimerization, and binds to androgen response elements (AREs) in target genes to regulate gene transcription.
• Upon binding of androgens to AR, activation of second messenger signaling pathways can also occur, regulating the transcription and translation of target genes.
• Abnormalities in the AR signaling pathway are associated with the development of various tumors, such as prostate cancer, bladder cancer, liver cancer, kidney cancer, lung cancer, among others.

Protein Expression

• AR is mainly expressed in tissues targeted by androgens, with high expression levels in male reproductive organs such as seminal vesicles, epididymis, testes, and prostate.
• Human Androgen Receptor isoform 2 (P10275-2): mainly expressed in the heart and skeletal muscle.
• Human Androgen Receptor isoform 3 (P10275-3): expressed in prostate basal and stromal cells (protein level).

Protein Localization

• Nucleus, cytoplasm.

Figure 2: ICC experimental result of Androgen Receptor protein, using Anti-Androgen Receptor antibody (ab108341). Green: Androgen Receptor, Red: alpha Tubulin, Blue: DAPI.

Isoforms and post-translational modifications

• Human (P10275): Isoform 1: 99 kDa (predicted); Isoform 2: 45 kDa (predicted)
• Isoforms 3-4: 67-68 kDa (predicted)
• Mouse (P19091): 99 kDa (predicted)
• Rat (P15207): 99 kDa (predicted)
• Ubiquitination
• Phosphorylation
• Palmitoylation

WB experiment tips

Precautions

• When using human samples for WB experiments, multiple bands may be detected for Androgen Receptor because human Androgen Receptor contains multiple isoforms and post-translational modifications.
• Please note that the expression levels of target proteins may vary in different samples, and it is necessary to confirm the expression levels of target proteins before testing. We recommend using the samples used in the antibody product manual as positive controls.
• Androgen Receptor lysate is prone to degradation after freezing, so it is recommended to use fresh lysate in WB experiments and complete sample preparation and gel running on the same day to avoid fragmented bands.
• If no bands/weak bands/non-specific bands appear after using RIPA lysate for certain samples, you can try using the 1% SDS heat lysate preparation method.
• If the expression level of Androgen Receptor in the sample to be tested is low, you can try to appropriately extend the exposure time during development.

Positive control

• Human LNCaP whole cell lysate
• Mouse testicular tissue lysate
• Rat testicular tissue lysate

Negative control

• Human PC3 whole cell lysate
• Mouse liver tissue lysate
• Rat liver tissue lysate

Example of Results

Figure 3: WB-Anti-Androgen Receptor (EP670Y) antibody product (ab52615).

Lane 1: Mouse testis tissue lysate (20 µg).
Lane 2: Rat testis tissue lysate (20 µg).
Lane 3: Mouse liver tissue lysate (20 µg).
Lane 4: Rat liver tissue lysate (20 µg).

Predicted band size: 98 kDa
Detected band size: 110 kDa

Figure 4: WB-Anti-Androgen Receptor (EPR1535(2)) antibody product (ab133273).

Lane 1: LNCaP (human prostate cancer epithelial cells) whole cell lysate (RIPA lysis).
Lane 2: LNCaP (human prostate cancer epithelial cells) whole cell lysate (1% SDS heat lysis).
Lane 3: 22Rv1 (human prostate cancer epithelial cells) whole cell lysate (RIPA lysis).
Lane 4: 22Rv1 (human prostate cancer epithelial cells) whole cell lysate (1% SDS heat lysis).

Predicted band size: 98 kDa
Detected band size: 120 kDa

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a sufficient amount of composite protease inhibitor to avoid degradation of the target protein.
2. Select a suitable lysis buffer to enrich more target proteins.
3. Keep the sample on ice throughout the sample preparation process.
4. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.
2. For target proteins with larger molecular weight, we recommend using a lower concentration separation gel for electrophoresis.

Transfer:

1. For target proteins with larger molecular weight, it is recommended to add SDS to a final concentration of 0.1% in the transfer buffer.
2. For target proteins with larger molecular weight, it is recommended to use a PVDF membrane with a pore size of 0.45 μm.
3. For target proteins with larger molecular weight, it is recommended to use 10% methanol or lower concentration in the transfer buffer.
4. We recommend using Ponceau S staining after transfer to confirm the success of the transfer (if fluorescence labeling detection is chosen, make sure to thoroughly clean the Ponceau S).

Blocking:

1. There is no blocking solution suitable for all systems, please choose the appropriate blocking solution.

Reference

1. Lu NZ, Wardell SE, Burnstein KL, et al. International Union of Pharmacology. LXV. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors. Pharmacol Rev. (2006). 58(4): 782‐797. doi: 10.1124/pr.58.4.9.
2. Koryakina Y, Ta HQ, Gioeli D. Androgen receptor phosphorylation: biological context and functional consequences. Endocr Relat Cancer. (2014). 21(4): T131‐T145. doi: 10.1530/ERC-13-0472.
3. Tan ME, Li J, Xu HE, Melcher K, Yong E. Androgen receptor: structure, role in prostate cancer and drug discovery. Acta Pharmacol Sin. (2015). 36(1):3‐23. doi: 10.1038/aps.2014.18.
4. 4. Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat Rev Cancer. (2015).15(12):701‐711. doi: 10.1038/nrc4016.