![<b>Up-regulation and Antibody-Peptide Competition</b> Extracts of CHO-T cells over-expressing the human insulin receptor unstimulated (5) or stimulated with 100 nM insulin for 10 min at 37°C (1-4) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the IR/IGF1R [pY1158] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphorylated peptide corresponding to the phosphopeptide immunogen (2), a generic phosphotyrosine-containing peptide (3), or the phosphopeptide immunogen (4). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignalTM method. The data show that only the phosphopeptide corresponding to IR/IGF1R [pY1158] completely blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the up-regulation of this site upon stimulation with insulin in this cell system.](/media/images/0096343.jpeg)
BP7096 CD220 + CD222 pTyr1158 antibody
see related secondary antibodiessee all 8 CD220 + CD222 products
0.1 ml / US$ 515
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 CD220 + CD222
Synonyms
IR, INSR, IGF-I receptor, IGFR, ,
Product review
Please read our product review about CD220 + CD222: Antibody Panel to Insulin Receptor (CD220).
Product Description
Rabbit anti CD220 + CD222 , Presentation: Aff - Purified. Product is tested for Frozen sections ( C ), Western blot / Immunoblot ( WB )
Properties
| Applications | Frozen sections ( C ), Western blot / Immunoblot ( WB ) |
| Reactivity | Human ( Hu ), Mouse ( Ms ), Rat ( Rt ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7096 |
| Swiss Prot. No. | P08069 |
| Quantity | 0.1 ml |
| Price | US$ 515 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7096.pdf |
Datasheet Extract
| Background | Biological actions of insulin and IGF1 are mediated by their respective cell surface receptors, both of which are receptor tyrosine kinases that regulate multiple signaling pathways through activation of a series of phosphorylation cascades. The IR and IGF1R are heterotetrameric proteins consisting of two ligand-binding α subunits and two β subunits that each contain a tyrosine kinase domain. Insulin/IGF1 binding to the extracellular domain leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. These two receptors differ in sequence in regions that confer specificity for the designated ligand as well as in certain intracellular signaling domains. These differences allow insulin and IGF-1 to regulate different physiological functions through receptors that share a very similar structure. Phosphorylation sites that are unique to each receptor presumably play a key role in these signaling differences. The catalytic loops within the tyrosine kinase domains of the IR/IGF1R contain a three tyrosine motif corresponding to Tyr1158, 1162 and 1163 (for the IR) and Tyr1131, 1135 and 1136 (for the IGF1R). It is generally believed that autophosphorylation within the activation loop proceeds in a processive manner initiating at the second tyrosine (1162 or 1135), followed by phosphorylation at the first tyrosine (1158 or 1131), then the last (1163 or 1136), upon which the IR or IGF1R becomes fully active. |
| Immunogen | Chemically synthesized phosphopeptide derived from the region of IR/IGF1R that contains tyrosine 1158 of the human insulin receptor (IR) as numbered according to Ebina, et al. (1146 according to Ullrich, et al.). Swiss Num.: P08069 Remarks: The corresponding residue in the IGF1R is 1131. The sequence is conserved in mouse and rat for both the IR and IGF1R. |
| Format | State: Liquid 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 insulin/insulin-like growth factor-1 receptor (IR/IGF1R). The final product is generated by affinity chromatography using an IR/IGF1R-derived peptide that is phosphorylated at tyrosine 1158 (tyrosine 1131 for IGF1R). BufferSystem: Dulbecco's phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), 50% glycerol with 1.0 mg/mL BSA (IgG, protease free) as a carrier, containing 0.05 % sodium azide as preservative. |
| Applications | Western blot (1:1000 starting dilution).
Positive Controls Used: CHO-T transfected with a vector containing insulin receptor and stimulated with insulin. |
| Specificity | This antibody detects IR/IGF1R. Species: Human, Mouse, Rat. |
| Storage | Store the antibody at -20 °C. Can be shipped at 2 - 8 °C.
Avoid repeated freezing and thawing. Centrifuge vial before opening. Shelf life: One year from despatch. |
| References | Motley, E.D., et al. (2003) Insulin-induced Akt activation is inhibited by angiotensin II in the vasculature through protein kinase C-alpha. Hypertension 41(3 Pt 2):775-780.
Wick, K.R., et al. (2003) Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor. J. Biol. Chem. 278(10):8460-8467. Motley, E.D., et al. (2002) Lysophosphatidylcholine inhibits insulin-induced Akt activation through protein kinase C-alpha in vascular smooth muscle cells. Hypertension 39(2 Pt 2):508-512. Hers, I., et al. (2002) Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes. Biochem. J. 368(Pt 3):875-884. Pender, C., et al. (2002) Regulation of insulin receptor function by a small molecule insulin receptor activator. J. Biol. Chem. 277(46):43565-43571. |
| 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 a 1:1000 starting dilution in Tris buffered saline supplemented with 3% Ig-free BSA and 0.1% Tween 20 overnight at 4°C or for two hours 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 The specificity of a Phosphorylation Site Specific Antibody (PSSA) in each experimental system can be confirmed through peptide competition. In this technique, aliquots of antibody are pre-incubated with peptide containing the sequence of the phosphopeptide immunogen used to raise the PSSA and the corresponding non-phosphopeptide. Following preincubation with the peptide, each antibody preparation is then used as a probe in antibody-based detection methods, such as Western blotting, immunocytochemistry, flow cytometry, or ELISA. With a PSSA specific for the phosphorylated target protein, pre-incubation with an excess of peptide containing the sequence of the phosphopeptide immunogen will block all antigen binding sites, while pre-incubation with the corresponding non-phosphopeptide will not affect the antibody. In performing the Peptide Competition Experiment, it is important to note that the optimal dilutions of both antibody and peptide should be determined empirically for each specific application. The optimal dilution of antibody in these procedures is below saturating, as determined by previous experiments in your system. The optimal dilution of peptide used in these procedures will depend on the overall affinity or avidity of the antibody, as well as the quantity of the target antigen. A 50-150 fold molar excess of peptide to antibody is found to be effective for most peptide competition experiments. In the example presented below, the PSSA is used as a dilution of 1:1000 and the peptides are used at a concentration of 333 nM. The total volume of the phosphopeptide and nonphosphopeptide pre-incubated antibody preparations is 2 mL, sufficient for probing Western blot strips, as well as for use in other antibody-based detection methods. Under these conditions, the molar excess of peptide to antibody is > / = 50. Procedure: 1. Prepare three identical test samples, such as identical PVDF or nitrocellulose strips to which the protein of interest has been transferred. The test samples should be blocked using a blocking buffer, such as Tris buffered saline supplemented with 0.1% Tween 20, and either 5% BSA or 5% non-fat dried milk. 2. Prepare 6.5 mL of working antibody stock solution (1:1000 in this example) by adding 6.5 μL of antibody stock solution to 6.5 mL of buffer containing blocking protein, such as TBS supplemented with 0.1% Tween 20, and either 3% BSA or 3% non-fat dried milk. 3. Apportion the unused PSSA into working aliquots and store at -20°C for future use (the stock PSSA contains 50% glycerol and will not freeze at this temperature). 4. Allow the lyophilized control peptides to reach room temperature, ideally under desiccation. 5. Reconstitute each of the control peptides to a concentration of 66.7 µM with nanopure water. (i.e. for a peptide with a molecular mass of 1500, reconstitution with 1 mL water yields a solution with a concentration of 66.7 µM). 6. Apportion the unused reconstituted peptide solutions into working aliquots and store at -20°C for future use. 7. Label 3 test tubes as follows: - tube 1: water only no peptide control - tube 2: phosphopeptide - tube 3: non-phosphopeptide 8. Into each tube, pipette the following components - tube 1: 2 mL diluted PSSA solution plus 10 µL nanopure water - tube 2: 2 mL diluted PSSA solution plus 10 µL phosphopeptide - tube 3: 2 mL diluted PSSA solution plus 10 µL non-phosphopeptide 9. Incubate the three tubes for 30 minutes at room temperature with gentle rocking. During this incubation, the peptides have the chance to bind to the combining site of the antibody. 10. At the end of the incubation step, transfer the contents of each of the three tubes to clean reaction vessels containing one of the three identical test samples. For Western blotting strips: Incubate the strips with the pre-incubated antibody preparations for 1 hour at room temperature or overnight at 4°C. Wash each strip four times, five minutes each, to remove unbound antibody. Transfer each strip to a new solution containing a labeled secondary antibody [e.g., goat F(ab)2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab)2 anti-rabbit IgG horseradish peroxidase conjugate. Remove unbound secondary antibody by thorough washing, and develop the signal using your chemiluminescent reagents and instrumentation. The signal obtained with antibody incubated with the "Water Only, No Peptide Control" (Tube 1), represents the maximum signal in the assay. This signal should be eliminated by preincubation with the "Phosphopeptide" (Tube 2), while pre-incubation with the "Non-Phosphopeptide" (Tube 3) should not impact the signal. If the "Phosphopeptide" only partially eliminates the signal, repeat the procedure using twice the volume of water or peptide solutions listed in Step 8. If partial competition is seen following pre-incubation with the "Non-Phosphopeptide", repeat the procedure using half the volumes of water or peptide solutions listed in Step 8. |
| Pictures | Up-regulation and Antibody-Peptide Competition Extracts of CHO-T cells over-expressing the human insulin receptor unstimulated (5) or stimulated with 100 nM insulin for 10 min at 37°C (1-4) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the IR/IGF1R [pY1158] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphorylated peptide corresponding to the phosphopeptide immunogen (2), a generic phosphotyrosine-containing peptide (3), or the phosphopeptide immunogen (4). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignalTM method. The data show that only the phosphopeptide corresponding to IR/IGF1R [pY1158] completely blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the up-regulation of this site upon stimulation with insulin in this cell system. |
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 'BP7096 CD220 + CD222'.
