![<b>Peptide Competition:</b> 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 β-arrestin-1 [pSer412] 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(TM) method.
<b>Phosphatase Stripping:</b>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 β-arrestin-1 pan antibody (5, 6), or 0.75 μg/mL β-arrestin-1 [pSer412] 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(TM) method. The data show that only the peptide corresponding to β-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 phospho-specific.](/media/images/0097326.jpeg)
BP7007 Beta-arrestin-1 (pSer412) antibody
see related secondary antibodiessee all 8 Beta-arrestin-1 products
0.1 ml / US$ 495
NOVUS BIOLOGICALS
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
Quick Overview
Rabbit anti Beta-arrestin-1 (pSer412) -
Synonyms
Arrestin beta-1, ARRB1, ARB1, ARR1, ,
Product review
Please read our product review about Beta-arrestin-1 (pSer412): Antibodies to Arrestin/beta-Arrestin Protein Family.
Product Description
Rabbit anti Beta-arrestin-1 (pSer412) -, Presentation: Aff - Purified. Product is tested for Western blot / Immunoblot ( WB )
Properties
| Applications | Western blot / Immunoblot ( WB ) |
| Reactivity | Rat ( Rt ), Mouse ( Ms ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7007 |
| Swiss Prot. No. | P29066 |
| Quantity | 0.1 ml |
| Price | US$ 495 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7007.pdf |
Datasheet Extract
| Polyclonal Antibody to Ã?-Arrestin-1 [pSer412] -Phosphospecific Antibody | |
| Concentration | 0.75 mg/ml |
| Background | β-arrestin-1 is a member of a family of proteins widely expressed but especially abundant in the central nervous system. Serving as an adaptor or scaffold molecule, β-arrestin-1 is essential for mitogenic signaling and mediates agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCRs, e.g., β2-adrenergic receptor). After binding to their ligand and interacting with heterotrimeric G proteins, GPCRs are phosphorylated by G-protein receptor kinases (GRKs) on serine residues. β-arrestin-1 in the cytosol is phosphorylated by ERK1&2 on serine 412 in a negative feedback mechanism and binds to the phosphorylated receptors at the plasma membrane. Serine 412 is then dephosphorylated and the GPCRs are internalized, leading to activation of the Ras - > Raf - > ERK1&2 signaling pathway. |
| Immunogen | The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of rat beta-arrestin-1 that contains serine 412.
Swiss Num.: P29066 |
| Format | State: Liquid purified Ig fraction Purification: Sequential epitope-specific chromatography.The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated ?-arrestin-1 protein. The final product is generated by affinity chromatography using a ?-arrestin-1-derived peptide that is phosphorylated at serine 412. BufferSystem: Dulbecco?s phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), with 1.0 mg/mL BSA (IgG, protease free) as a carrier, containing 0.05% sodium azide |
| Applications | Western blot. For Western blot applications, using the antibody at 0.1-1.0 μg/mL is recommended.
At 0.75 μg/mL, the dilution provides 100 mL working solution, which at 10 mL/blot allows 10 blots to be performed. Positive controls used: PC12 and CHO-K cells. |
| Specificity | The antibody recognizes mouse and rat β-arrestin-1.
Human and cow (77% homologous) β-arrestin-1 have not been tested, but are expected to react. |
| Storage | Store in aliquots at -80°C.
Avoid repeated freezing and thawing. Shelf life: one year from despatch. |
| References | 1. 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. 2. Miller, W.E., et al. (2000) beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis. J. Biol.Chem. 275(15):11312-11319. 3. Lefkowitz, R.J. (1998) G protein-coupled receptors. III. New roles for receptor kinases and betaarrestins in receptor signaling and desensitization. J. Biol. Chem. 273(30):18677-18680. |
| Protocols | Western Blotting Procedure
1. Lyse approximately 10e7 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application. 2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifugedat 100,000 x g for 30 minutes for greater clarification. 3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Polypropylene tubes are recommended for storing cell lysates. 4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100°C. 5. Load 10-30 µg of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by SDS-PAGE. 6. In preparation for the Western transfer, cut a piece of PVDF membrane slightly larger than the gel. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. Alternatively, nitrocellulose may be used. 7. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes. 8. Assemble the gel and membrane into the sandwich apparatus. 9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature. 10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes. 11. Block the membrane with blocking buffer (formulation provided below) for one hour at room temperature or overnight at 4°C. 12. Incubate the blocked blot with primary antibody at 0.1-1.0 µg/ml in Tris buffered saline supplemented with 3% BSA and 0.1% Tween 20 overnight at 4°C or for one hour at room temperature. 13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20. 14. Detect the antibody band using an appropriate secondary antibody, such as goat F(ab')2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab')2 anti-rabbit IgG horseradish peroxidase conjugate in conjunction with your chemiluminescence reagents and instrumentation. Cell Lysis Buffer Formulation: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM Na3VO4 0.1% SDS 0.5% sodium deoxycholate 1% Triton-X 100 10% glycerol 1 mM PMSF (made from a 0.3 M stock in DMSO) or 1 mM AEBSF (water soluble version of PMSF) 60 µg/mL aprotinin 10 µg/mL leupeptin 1 µg/mL pepstatin (alternatively, protease inhibitor cocktail such as Sigma Cat. # P2714 may be used) Transfer Buffer Formulation: 2.4 gm Tris base 14.2 gm glycine 200 mL methanol Q.S. to 1 liter, then add 1 mL 10% SDS. Cool to 4°C prior to use. Tris Buffered Saline Formulation: 20 mM Tris-HCl, pH 7.4 0.9% NaCl Blocking Buffer Formulation: 100 mL Tris buffered saline 5 gm BSA 0.1 mL Tween 20 Peptide Competition Experiment Procedure: To demonstrate the specificity of a Phosphorylation Site Specific Antibody (PSSA), we recommend the following peptide competition experiment: The control peptides used have the sequences of the phosphopeptide immunogen used to raise the antibody and the corresponding non-phosphorylated peptide. In the competition experiment, 200-500 fold molar excess of the phosphorylated and non-phosphorylated peptides are pre-incubated with aliquots of the antibody prior to use in immunoassay procedures. A sample calculation for the determination of the 200 fold molar excess of peptide to antibody is presented below. The following assumptions have been made: -The molecular mass of an IgG molecule is 150,000 daltons. -Each mole of antibody binds two moles of peptide. -The Phosphorylation Site Specific Antibody is used at a concentration of 0.5 μg/mL. The optimal antibody concentration for use in peptide competition experiments is below saturating as determined by previous experiments in your system. A final antibody concentration of 0.5 μg/mL is satisfactory for most applications. The molarity of the 0.5 μg/mL antibody solution is: (0.5 μg/mL)(1000 mL/L)/(150,000 μg/μmole) = 0.00333 μM. Because each mole of antibody binds two moles of peptide, 0.5 μg/mL antibody can bind 0.00667 μM of peptide. A 200 fold molar excess of peptide is (200)(0.00667 μM) = 1.334 μM. The following procedure describes peptide competition experiments using antibody at a concentration of 0.5 μg/mL and a 200 fold molar excess of peptides based on the calculation above, in a total volume of 2 mL. Procedure: 1. Prepare three identical test samples, such as identical nitrocellulose or PVDF strips with transferred protein. The test samples should be blocked with BSA or non-fat dried milk in a buffer compatible with an antibody based detection method, such as Tris buffered saline or phosphate buffered saline. 2. Slowly thaw the Phosphorylation Site Specific Antibody on ice. 3. Prepare 3 mL of a 2x (1 μg/mL) antibody stock solution in a buffer appropriate for the application. Suggested buffer formulations are TBS or PBS supplemented with blocking protein such as BSA or non-fat dried milk. 4. Apportion the unused Phosphorylation Site Specific Antibody into working aliquots and store at -80°C for future use. 5. The lyophilized control peptides should be warmed to room temperature, ideally under desiccation. 6. Reconstitute each of the control peptides to a concentration of 100 μM using nanopure water at room temperature. As indicated on the peptide labels, each vial contains 0.1 mg. For a peptide with a molecular mass of 1500, reconstitution with 0.67 mL water yields a solution with a concentration of 100 μM. 7. Allow the peptides to dissolve at room temperature, then gently triturate several times using a pipette. Avoid introducing air bubbles. 8. Label 3 test tubes as follows: -tube 1: water only no peptide control -tube 2: phosphopeptide -tube 3: non-phosphopeptide 9. Prepare 2x peptide stock solutions (2.66 μM) or water control by pipetting the following: -tube 1: water control stock: 0.973 mL buffer (TBS or PBS plus blocking protein) plus 27 μL water. -tube 2: phosphopeptide 2x stock: 0.973 mL buffer (TBS or PBS plus blocking protein) plus 27 μL reconstituted (100 μM) phosphopeptide. -tube 3: non-phosphopeptide 2x stock: 0.973 mL buffer (TBS or PBS plus blocking protein) plus 27 μL reconstituted (100 μM) non-phosphopeptide. 10. Apportion unused reconstituted peptide solutions into working aliquots and store at -20°C for future use. 11. Pipette 1 mL of the 2x antibody stock into each of the tubes marked 1, 2, and 3. The tubes should be incubated for 30 minutes at room temperature with gentle rocking. 12. The pre-incubated antibody in each of the three tubes is then ready for use. Pipette the contents of each tube onto the three identical test samples. For Western blotting strips: -Incubate these strips for 2 hours at room temperature, followed by several washes to remove unbound antibody. -Transfer each strip to a new solution containing a labeled secondary antibody (example goat anti-rabbit IgG-alkaline phosphatase conjugate). -Remove unbound secondary antibody by thorough washing and develop bands. The signals obtained with antibody incubated with "(1) water only no peptide control", which represents the maximum signal, and the signals obtained with "(2) phosphopeptide" and "(3) non-phosphopeptide" are readily compared under these conditions. |
| Pictures | 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 β-arrestin-1 [pSer412] 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(TM) 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 β-arrestin-1 pan antibody (5, 6), or 0.75 μg/mL β-arrestin-1 [pSer412] 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(TM) method. The data show that only the peptide corresponding to β-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 phospho-specific. |
12 Secondary Antibodies
| Catalog No. | Name / Host | Presentation | Reactivity | ||||
|---|---|---|---|---|---|---|---|
| R1364B | Rabbit IgG (H&L) | ||||||
| Goat | Biotin | 2 mg / US$ 295 | |||||
| R1364F | Rabbit IgG (H&L) | ||||||
| Goat | FITC | 2 mg / US$ 285 | |||||
| R1364T | Rabbit IgG (H&L) | ||||||
| Goat | TRITC | 2 mg / US$ 285 | |||||
| R1364TR | Rabbit IgG (H&L) | ||||||
| Goat | Texas Red | 2 mg / US$ 295 | |||||
| R1364HRP | Rabbit IgG (H&L) | ||||||
| Goat | HRP | 2 mg / US$ 295 | |||||
| R1364AP | Rabbit IgG (H&L) | ||||||
| Goat | AP | 1 mg / US$ 325 | |||||
| R1458C2 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy2 | 1 mg / US$ 445 | |||||
| R1458C3 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3 | 1 mg / US$ 445 | |||||
| R1458C35 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3.5 | 1 mg / US$ 445 | |||||
| R1458C5 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5 | 1 mg / US$ 445 | |||||
| R1458C55 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5.5 | 1 mg / US$ 445 | |||||
| R1427R | Rabbit IgG (H&L) F(ab')2 Fragment multi-species ads. | ||||||
| Donkey | PE | 1 ml / US$ 455 | |||||
Click here to see all secondary antibodies for 'BP7007 Beta-arrestin-1 (pSer412)'.