![Peptide Competition:
Extracts prepared from CEF cells expressing FAK and plated on fibronectin
were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with FAK [pY397] 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 phosphotyrosine-containing peptide (3), a phosphopeptide derived
from the corresponding region of Pyk2 (4), or, the phosphopeptide immunogen
(5). After washing, membranes were incubated with goat F(ab)2 anti-rabbit
IgG HRP-conjugate and bands were detected using the Pierce
SuperSignal(TM) method.
The data show that only the peptide corresponding to FAK [pY397] blocks the
antibody signal, thereby demonstrating the specificity of the antibody.](/media/images/0094190.jpeg)
BP7069 Focal Adhesion Kinase / FAK (pTyr397) antibody
see related secondary antibodiessee all 62 Focal Adhesion Kinase / FAK products
0.1 ml / please contact our distributor
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 Focal Adhesion Kinase / FAK (pTyr397) -
Synonyms
Protein-tyrosine Kinase 2 FADK1 FAK1 PTK2 PP125FAK
Product review
Please read our product review about Focal Adhesion Kinase / FAK (pTyr397) : Antibodies to Focal Adhesion Kinase (FAK).
Product Description
Rabbit anti Focal Adhesion Kinase / FAK (pTyr397) -, Presentation: Aff - Purified. Product is tested for Frozen sections ( C ), Immunofluorescence ( IF ), Westernblot / Immunoblot ( WB )
Properties
| Applications | Frozen sections ( C ), Immunofluorescence ( IF ), Westernblot / Immunoblot ( WB ) |
| Reactivity | Human ( Hu ), Mouse ( Ms ), Rat ( Rt ), Chicken ( Chk ), Xenopus ( Xen ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7069 |
| Swiss Prot. No. | Q05397 |
| Quantity | 0.1 ml |
| Price | please contact our distributor |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7069.pdf |
Datasheet Extract
| Polyclonal Antibody to FAK [pTyr397] Phosphospecific Antibody | |
| Background | Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor protein tyrosine kinase that was discovered as a substrate for Src, and is a key element of integrin signaling. FAK plays a central role in cell spreading, differentiation, migration, cell death and acceleration of the G1 to S phase transition of the cell cycle. Tyrosine 397 is the autophosphorylation site of FAK, and involved in its initial activation. The phosphorylated site binds Src family SH2 domains and the p85 subunit of PI3-Kinase, and activates cell migration and invasion. |
| Immunogen | The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human FAK that contains tyrosine 397. Swiss Num.: Q05397 Remarks: The sequence is conserved in mouse, rat, chicken and frog. |
| Format | State: Liquid 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 FAK. The final product is generate 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 and 0.05% sodium azide as preservative |
| Applications | Western blotting: use a 1:1000 starting dilution. Positive controls used: Chick embryo fibroblasts expressing FAK protein and plated on fibronectin, and NIH3T3 cells treated with PDGF.
Previous lots of this antibody have been used in immunocytochemistry and immunohistochemistry. |
| Specificity | The antibody recognizes FAK (pY397). Species: Human, mouse, chicken, frog, and fly. |
| Storage | Can be shipped at 2-8°C. upon arrival, centrifuge before opening to settle vial contents.
Then aliquot and store at -20°C. Avoid repeated freezing and thawing. Shelf life: one year from despatch. |
| References | Bock, H.H. and J. Herz (2003) Reelin activates SRC family tyrosine kinases in neurons. Curr. Biol. 13(1):18-26 Fernandis, A.Z., et al. (2003) Differential regulation of CXCR4-mediated T-cell chemotaxis and mitogenactivated protein kinase activation by the membrane tyrosine phosphatase, CD45. J. Biol. Chem. 278(11):9536-9543 Grace, E.A. and J. Busciglio (2003) Aberrant activation of focal adhesion proteins mediates fibrillar amyloid beta-induced neuronal dystrophy. J. Neurosci. 23(2):493-502
Hsia, D.A., et al. (2003) Differential regulation of cell motility and invasion by FAK. J. Cell Biol. 160(5):753-767 Pankov, R., et al. (2003) Specific beta1 integrin site selectively regulates Akt/protein kinase B signaling via local activation of protein phosphatase 2A. J. Biol. Chem. 278(20):18671-18681 Cary, L.A., et al. (2002) SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading. Mol. Cell. Biol. 22(8):2427-2440 Eliceiri, B.P., et al. (2002) Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling. J. Cell Biol. 157(1):149-160 Hauck, C.R., et al. (2002) v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrincontaining invadopodia promotes cell invasion. J. Biol. Chem. 277(15):12487-12490 |
| 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. This cell lysis buffer formulation is available as a separate product which requires supplementation with protease inhibitors immediately prior to use. 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 ultracentrifuged at 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. 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) overnight at 4°C. 12. Incubate the blocked blot with primary antibody at a dilution of 1:1,000 in Tris buffered saline supplemented with 1% Ig-free BSA and 0.1% Tween 20 overnight at 4°C or 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 catalog number 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 Phosphorylation Site Specific Antibodies (PSSAs) have been developed to enable the specific and sensitive detection of phosphorylation of particular amino acid residues in target proteins, while circumventing the need for protein purification, phosphopeptide mapping or handling radioactivity. The specificity of a 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. There have been developed a line of control peptides specifically for use in peptide competition experiments with our PSSAs. These peptides, available as separate catalog items, are provided in pairs which contain the sequences of the phosphopeptide immunogen and the corresponding non-phosphopeptide. 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. If an optimal antibody dilution has not been determined in your system, please refer to the Suggested Working Dilution on the antibody Product Analysis Sheet for guidance on an appropriate starting dilution. 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 non-phosphopeptide-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: 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. 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. 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). Allow the lyophilized control peptides to reach room temperature, ideally under desiccation. Reconstitute each of the control peptides Label 3 test tubes as follows: - tube 1: water only no peptide control - tube 2: phosphopeptide - tube 3: non-phosphopeptide 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 pre-incubation 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 | Peptide Competition:
Extracts prepared from CEF cells expressing FAK and plated on fibronectin were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with FAK [pY397] 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 phosphotyrosine-containing peptide (3), a phosphopeptide derived from the corresponding region of Pyk2 (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab)2 anti-rabbit IgG HRP-conjugate and bands were detected using the Pierce SuperSignal(TM) method. The data show that only the peptide corresponding to FAK [pY397] blocks the antibody signal, thereby demonstrating the specificity of the antibody. |
12 Secondary Antibodies
| Catalog No. | Name / Host | Presentation | Reactivity | ||||
|---|---|---|---|---|---|---|---|
| R1364B | Rabbit IgG (H&L) | ||||||
| Goat | Biotin | 2 mg / 170,00 € | |||||
| R1364F | Rabbit IgG (H&L) | ||||||
| Goat | FITC | 2 mg / 160,00 € | |||||
| R1364T | Rabbit IgG (H&L) | ||||||
| Goat | TRITC | 2 mg / 160,00 € | |||||
| R1364TR | Rabbit IgG (H&L) | ||||||
| Goat | Texas Red | 2 mg / 170,00 € | |||||
| R1364HRP | Rabbit IgG (H&L) | ||||||
| Goat | HRP | 2 mg / 170,00 € | |||||
| R1364AP | Rabbit IgG (H&L) | ||||||
| Goat | AP | 1 mg / 200,00 € | |||||
| R1458C2 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy2 | 1 mg / 320,00 € | |||||
| R1458C3 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3 | 1 mg / 320,00 € | |||||
| R1458C35 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3.5 | 1 mg / 320,00 € | |||||
| R1458C5 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5 | 1 mg / 320,00 € | |||||
| R1458C55 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5.5 | 1 mg / 320,00 € | |||||
| R1427R | Rabbit IgG (H&L) F(ab')2 Fragment multi-species ads. | ||||||
| Donkey | PE | 1 ml / 330,00 € | |||||
Click here to see all secondary antibodies for 'BP7069 Focal Adhesion Kinase / FAK (pTyr397) '.
