discontinued

BP7219 Src Family Negative Regulatory [pTyr] Site antibody

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0.1 ml / €420.00

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Rabbit anti Human, Mouse, Rat Src Family Negative Regulatory [pTyr] Site

BP7219

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  • BP7219

Product Description for Src Family Negative Regulatory [pTyr] Site

Rabbit anti Human, Mouse, Rat Src Family Negative Regulatory [pTyr] Site.
Presentation: Aff - Purified
Product is tested for Western blot / Immunoblot.

Properties for Src Family Negative Regulatory [pTyr] Site

Product Category Primary Antibodies
Quantity 0.1 ml
Presentation Aff - Purified
Reactivity Hu, Ms, Rt
Applications WB
Clonality Polyclonal
Host Rabbit
Shipping to Worldwide
PDF datasheet View Datasheet
Manufacturer Acris Antibodies GmbH
Material safety datasheet MSDS for Polyclonal Antibodies (de)

Datasheet Extract

Immunogen
Immunogen:
Chemically synthesized phosphopeptide derived from the region of human Hck that contains tyrosine 522.
Remarks:
The sequence is conserved in mouse and rat, and also among other members of the Src protein family.
Application Western blot (0.35-1.0 μg/ml; at 0.50 μg/mL, the dilution provides 100 mL working solution, which at 10 mL/blot allows 10 blots to be performed).
Positive Controls Used: PC12 cells +/- sorbitol, Chick Embryo Fibroblast (CEF) cells expressing Src protein and plated on fibronectin, and recombinant activated Fyn, Lck and Lyn proteins.
Background The Src family of non-receptor tyrosine kinases is involved in the modulation of a wide variety of normal and pathogenic cellular processes including growth, development, differentiation, and carcinogenesis. These proteins contain a consensus structure comprised of one or more N-terminal SH2 and/or SH3 domains involved in protein-protein interactions, a central catalytic tyrosine kinase domain, and a short C-terminal domain. This C-terminal domain contains a tyrosine residue that, when phosphorylated by Csk tyrosine kinase, functions as a negative regulatory site (tyrosines 530, 531, 522, 505, and 508 in Src, Fyn, Hck, Lck, and Lyn, respectively) by providing a binding site for the N-terminal SH2 domain. The association of these two domains blocks access to a critical tyrosine residue in the activation loop, the phosphorylation of which is required for enzyme activation. When the C-terminal domain tyrosine residue is dephosphorylated, the catalytic domain tyrosine can be maximally phosphorylated leading to full kinase activation.
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 concentration of 0.1-1.0 μg/mL in Tris buffered saline supplemented with 3% BSA and 0.1% Tween 20 for 2 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 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.
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.
General Readings Yasuda, K., et al. (2002) Fyn is essential for tyrosine phosphorylation of Csk-binding protein/phosphoprotein associated with glycolipid-enriched microdomains in lipid rafts in resting T cells. J. Immunol. 169(6):2813-2817.
Young, M.A., et al. (2001) Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation. Cell 105(1):115-126.
Hubbard, S.R., and J.H. Till (2000) Protein tyrosine kinase structure and function. Ann. Rev. Biochem. 69:373-398.
Egan, C., et al. (1999) Activation of Src in human breast tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530. Oncogene 18(5):1227-1237.
Thomas, S.M. and J.S. Brugge (1997) Cellular functions regulated by Src family kinases. Ann. Rev. Cell Devel. Biol. 13:513-609.
Storage Store at -80 şC. Upon initial thawing, aliquot and store at -80 şC. Avoid repeated freezing and thawing.
Shelf life: one year from despatch.
Format
Purification:
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 Hck protein. The final product is generated by affinity chromatography using a Hck-derived peptide that is phosphorylated at tyrosine 522.
Buffer System:
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 as preservative
State:
Liquid Ig fraction
Aff - Purified
Specificity
Specificity:
This antibody detects Src kinase family members (Src, Fyn, Lck, Lyn, and Hck).
Species:
Human, Rat, Mouse.

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