Antibodies to Arrestin/beta-Arrestin Protein Family - FocusOn 076

Introduction

Vision involves the conversion of light into electrochemical signals that are processed by the retina and subsequently sent to and interpreted by the brain. The process of converting light to an electrochemical signal begins when the membrane-bound protein, rhodopsin, absorbs light within the retina. Photoexcitation of rhodopsin causes the cytoplasmic surface of the protein to become catalytically active.

In the active state, rhodopsin activates transducin, a GTP binding protein. Once activated, transducin promotes the hydrolysis of cGMP by phosphodiesterase (PDE). The decrease of intracellular cGMP concentrations causes the ion channels within the outer segment of the rod or cone to close, thus causing membrane hyperpolarization and, eventually, signal transmission. Rhodopsin’s activity is believed to be shut off by its phosphorylation followed by binding of the soluble protein Arrestin.

Arrestins are cytosolic proteins that are involved in G protein-coupled receptor (GPCR) desensitization. Arrestin binding to activated GPCRs is phosphorylation dependent and, once bound, uncouple the GPCR from the associated heterotrimeric G proteins. There are currently 4 known mammalian isoforms, beta-arrestin1 (arrestin2), beta-arrestin2 (arrestin3), visual arrestin (arrestin1), and cone arrestin (X-arrestin) The beta-isoforms are ubiquitously expressed and are known to interact with acetylcholine and adrenergic receptors. Visual and cone arrestins are found to interact directly with transducin. S-arrestin, also known as S-antigen, is a major soluble photoreceptor protein that is involved in desensitization of the photoactivated transduction cascade. It is expressed in the retina and the pineal gland and inhibits coupling of rhodopsin to transducin in vitro. Additionally, S-arrestin is highly antigenic, and is capable of inducing experimental autoimmune uveoretinitis. Mutations in this gene have been associated with Oguchi disease, a rare autosomal recessive form of night blindness.

Antibody Panel to Arrestin/beta-Arrestin Protein Family

Acris Antibodies a range of antibodies for the detection of visual arrestin (S-arrestin), pan Arrestin (isoforms beta-arrestin 1 and 2), one phosphospecific antibody which was produced against a synthesized phosphopeptide derived from the region of rat ß-arrestin-1 that contains serine 412 as well as its phospho/non phospho control peptides.

<b>Picture 1:</b><br>Antibody BM4547 to S-arrestin (S-antigen) staining bovine retina (the rod outer section) at 1:100

Picture 1:
Antibody BM4547 to S-arrestin (S-antigen) staining bovine retina (the rod outer section) at 1:100

<b>Peptide Competition:</b><br>Extracts prepared from PC12 cells were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4oC, then were incubated with 0.75 µg/mL ß-arrestin-1 [pS412] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phospho-serine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method.<br><br><b>Phosphatase Stripping:</b><br>Extracts and membranes were prepared as above. The membrane was either not treated (5, 7) or treated (6, 8) with PP2A phosphatase, then incubated with ß-arrestin-1 pan antibody (5, 6), or 0.75 µg/mL ß-arrestin-1 [pS412]antibody (7, 8) for two hours at room temperature. After washing,membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method.<br>The data show that only the peptide corresponding to ß-arrestin-1 [pS412] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal produced by the phospho antibody, but not the pan antibody, verifying that it is phosphospecific.

Peptide Competition:
Extracts prepared from PC12 cells were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4oC, then were incubated with 0.75 µg/mL ß-arrestin-1 [pS412] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phospho-serine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method.

Phosphatase Stripping:
Extracts and membranes were prepared as above. The membrane was either not treated (5, 7) or treated (6, 8) with PP2A phosphatase, then incubated with ß-arrestin-1 pan antibody (5, 6), or 0.75 µg/mL ß-arrestin-1 [pS412]antibody (7, 8) for two hours at room temperature. After washing,membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method.
The data show that only the peptide corresponding to ß-arrestin-1 [pS412] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal produced by the phospho antibody, but not the pan antibody, verifying that it is phosphospecific.

Antibody Panel to Arrestin/beta-Arrestin Protein Family

Visual arrestin (S-arrestin), pan Arrestin (isoforms beta-arrestin 1 and 2), Arrestin1, beta-arrestin antibody.

Literature

Deshpande DA, Theriot BS, Penn RB, Walker JK.{beta}-Arrestins specifically constrain {beta}2-adrenergic receptor signaling and function in airway smooth muscle.FASEB J. 2008 Mar 19 [Epub ahead of print].

Pfleger KD, Dalrymple MB, Dromey JR, Eidne KA.Monitoring interactions between G-protein-coupled receptors and beta-arrestins.Biochem Soc Trans. 2007 Aug;35(Pt 4):764-6.

Xiao K, McClatchy DB, Shukla AK, Zhao Y, Chen M, Shenoy SK, Yates JR 3rd, Lefkowitz RJ.Functional specialization of beta-arrestin interactions revealed by proteomic analysis.Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):12011-6. Epub 2007 Jul 9.

Miller, W.E., et al. (2000) Bea-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dpendent targeting of c-SRC in receptor endocytosis.J. Biol.Chem. 275(15):11312-11319.

Tohgo, A., et al. (2002) Beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation.J. Biol. Chem. 277(11):9429-9436.

Premont RT.(2005)Once and future signaling: g protein-coupled receptor kinase control of neuronal sensitivity.Neuromolecular Med.2005; 7(1-2):129-48.

12 Item(s)

per page

Primary Antibodies

Catalog No. Host Iso. Clone Pres. React. Applications  

Arrestin beta-1 / ARRB1 antibody

Immunohistochemistry (IHC) analyzes of Arrestin 1 antibody (Cat.-No.: AP06496PU-N) in paraffin-embedded human brain tissue. Rabbit Aff - Purified Hu ICC/IF, P, WB
0.1 mg / €350.00
  Acris Antibodies GmbH

Arrestin beta-1 / ARRB1 antibody

Western blot (WB) analysis of Arrestin-β-1 antibody (Cat.-No.: AP06791PU-N) at 1/500 dilution.Lane1: MCF-7 whole cell lysateLane2: Mouse brain tissue lysateLane3: H9C2 whole cell lysate Rabbit Aff - Purified Hu, Ms, Rt ICC/IF, P, WB
0.1 mg / €350.00
  Acris Antibodies GmbH

Arrestin beta-1 / ARRB1 antibody

Arrestin beta-1 / ARRB1 Rabbit IgG Aff - Purified Hu, Ms, Rt ICC/IF, P, WB
0.1 mg / €420.00
  Novus Biologicals Inc.

Arrestin beta-1 / ARRB1 antibody

Immunohistochemistry: B-Arrestin-1 Antibody (E274) [NB110-55485] - Immunohistochemical analysis of paraffin-embedded human lymph node using anti-ß-arrestin Rabbit IgG Purified Hu, Ms, Rt F, ICC/IF, P, WB
0.1 ml / €490.00
  Novus Biologicals Inc.

Arrestin beta-2 / ARRB2 antibody

Human Skin; ARRB2 antibody - middle region (ARP48007_P050) in Human Skin cells using Immunohistochemistry Rabbit Aff - Purified African clawed frog, Bov, Can, Chk, Hu, Ms, Por, Rb, Rt, Ze P, WB
50 µg / €350.00
  AVIVA Systems Biology

Arrestin beta-2 / ARRB2 antibody

Western Blot: Arrestin beta 2 Antibody [NB100-1515]-(0.3µg/ml) staining of Human Brain lysate (35µg protein in RIPA buffer). Goat Aff - Purified Hu, Ms, Rt E, IP, P, WB
0.1 mg / €400.00
  Novus Biologicals Inc.

Arrestin beta-2 / ARRB2 antibody

Western Blot: Arrestin Antibody [NB300-587] - Figure 1 shows a Western blot of pan Arrestin in rat retina using NB300-587. Rabbit Aff - Purified Bov, Rt IP, WB
0.1 mg / €400.00
  Novus Biologicals Inc.

Arrestin C / ARR3 antibody

Western Blot: Arrestin-C Antibody [NBP1-19629] - extracts from LOVO cells. Rabbit Aff - Purified Hu ICC/IF, P, WB
0.1 mg / €420.00
  Novus Biologicals Inc.

S-arrestin / SAG antibody

S-arrestin / SAG Mouse IgG1 PDS-1 Biotin Bov, Hu, Por C, ICC/IF, P, WB
0.1 ml / €360.00
  Novus Biologicals Inc.

S-arrestin / SAG antibody

S-arrestin / SAG Mouse IgG1 PDS-1 DyLight488 Bov, Hu, Por C, ICC/IF, P, WB
0.1 ml / €390.00
  Novus Biologicals Inc.

S-arrestin / SAG antibody

S-arrestin / SAG Mouse IgG1 PDS-1 HRP Bov, Hu, Por C, ICC/IF, P, WB
0.1 ml / €390.00
  Novus Biologicals Inc.

S-arrestin / SAG antibody

S-arrestin / SAG Mouse IgG1 PDS-1 Purified Bov, Hu, Por C, ICC/IF, P, WB
25 µl / €160.00
  Novus Biologicals Inc.

12 Item(s)

per page
  • LinkedIn