![<i>UP-REGULATION AND ANTIBODY-PEPTIDE COMPETITION</i> <b>Left panel:</b> Lysates from Jurkat cells untreated (lane 1) or treated with 10 µM hydrogen peroxide for 10 minutes (lanes 2-5) were resolved on a 10% Tris-glycine gel and transferred to PVDF. Membrane was blocked with a 3% milk-TBST buffer for one hour at room temperature, then incubated with the ETS1[pT38] antibody overnight at 4°C in a 1% milk-TBST buffer, following prior incubation with: no peptide (lanes 1, 2), the non-phosphopeptide corresponding to the immunogen (lane 3), a generic phosphothreonine-containing peptide (lane 4), or the phosphopeptide immunogen (lane 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 ETS1 [pT38] blocks the signal, verifying the specificity of the antibody. The data also show up - regulation of the signal upon hydrogen peroxide treatment in this cell system. <b>Right panel:</b> Extracts of Jurkat cells untreated (lane 1) or treated with hydrogen peroxide for ten minutes (lanes 2-5) that were either pretreated with the MEK inhibitor PD98059 (lane 3), the JNK inhibitor SP600125 (lane 4), or the p38 inhibitor SB202190 (lane 5). Western blotting was performed as described above using the ETS1[pT38] antibody or an antibody recognizing total ETS1 protein. The data illustrate regulation of this phosphorylation site by the ERK branch of the MAPK signaling cascade.](/media/images/0093100.jpeg)
BP7064 ETS1 (pT38) antibody
see related secondary antibodiessee all 22 ETS1 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 ETS1 (pT38)
Synonyms
ETS-1, p54, EWSR2, , Protein C-ets-1, v-ets Erythroblastosis Virus E26 Oncogene Homolog 1 (avian)
Product review
Please read our product review about ETS1 (pT38): Antibodies to ETS1 and ETS2 oncoproteins.
Product Description
Rabbit anti ETS1 (pT38) , Presentation: Aff - Purified. Product is tested for Western blot / Immunoblot ( WB )
Properties
| Applications | Western blot / Immunoblot ( WB ) |
| Reactivity | Human ( Hu ), Mouse ( Ms ), Rat ( Rt ), Rabbit ( Rb ), Chicken ( Chk ), Xenopus ( Xen ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7064 |
| Swiss Prot. No. | P14921 |
| Quantity | 0.1 ml |
| Price | US$ 515 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7064.pdf |
Datasheet Extract
| Background | The proto-oncogene ETS (E-twenty-six-specific sequence) is the founding member of a family of transcription factors that share a highly conserved DNA-binding domain, called the ETS domain, which recognizes a nucleotide motif with a GGAA/T core sequence. The ETS genes are dispersed on two separate chromosomal loci called ets-1, which codes for a 54 kDa protein, and ets-2, which codes for a 56 kDa protein. They are responsible for the regulation of critical genes involved in cell proliferation, differentiation, lymphoid development, motility, invasion, angiogenesis, and apoptosis. ETS proteins, which are highly expressed in T and B lymphoid lineages, are phosphorylated on threonine 38 by ERK1/2. Phosphorylation at this site allows an increase in the transactivational activity of the ETS protein. |
| Immunogen | The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human ETS1 that contains threonine 38. Swiss Num.: P14921 |
| Format | State: Liquid Ig fraction Purification: Sequential epitope-specific chromatography. The antibody has been negatively pre-adsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated ETS. The final product is generated by affinity chromatography using an ETS1-derived peptide that is phosphorylated at threonine 38. BufferSystem: Dulbeccos 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 a presevative |
| Applications | Western blotting: 1:1000.
Positive control used: Jurkat cells treated with hydrogen peroxide or PMA. |
| Specificity | Human ETS1. Chicken, frog (Xenopus laevis), mouse, rat, and rabbit (each 100% homologous) have not been tested, but are expected to react. |
| Storage | Can be shipped at 2-8°C. Aliquot and store at -20°C. Avoid repeated freezing and thawing.
Shelf life: one year from despatch. |
| References | Seidel, J.J. and B.J. Graves (2001) An ERK2 docking site in the Pointed domain distinguishes a subset of ETS transcription factors. Genes and Dev. 16:127-137.
Yordy, J.S. and R.C. Musise-Helmericks (2000) Signal transduction and the Ets family of transcription factors. Oncogene 19:6503-6513. Smith, J.L. et al. (2000) ets-2 is a target for Akt(protein kinase B)/jun N-terminal kinase signalling pathway in macrophages of motheaten-viable mutant mice. Mol. Cell. Biol. 20:8026-8034. Slupsky, C.M., et al. (1998) Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site. Proc. Nat'l. Acad. Sci. USA 95:12129-12134. Yang, B.-S., et al. (1996) Ras-mediated phosphorylation of a conserved threonine residue enhances the transactivation activities of c-Ets-1 and c-Ets-2. Mol. Cell. Biol. 16:538-547. |
| 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. Cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are 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 100oC. 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. 12. Incubate the blocked blot with primary antibody at a 1:1000 dilution in Tris buffered saline supplemented with 1% milk and 0.1% Tween 20 overnight at 4oC 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 4oC 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 3 gm non-fat dried milk 0.1 mL Tween 20 |
| Pictures | UP-REGULATION AND ANTIBODY-PEPTIDE COMPETITION Left panel: Lysates from Jurkat cells untreated (lane 1) or treated with 10 µM hydrogen peroxide for 10 minutes (lanes 2-5) were resolved on a 10% Tris-glycine gel and transferred to PVDF. Membrane was blocked with a 3% milk-TBST buffer for one hour at room temperature, then incubated with the ETS1[pT38] antibody overnight at 4°C in a 1% milk-TBST buffer, following prior incubation with: no peptide (lanes 1, 2), the non-phosphopeptide corresponding to the immunogen (lane 3), a generic phosphothreonine-containing peptide (lane 4), or the phosphopeptide immunogen (lane 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 ETS1 [pT38] blocks the signal, verifying the specificity of the antibody. The data also show up - regulation of the signal upon hydrogen peroxide treatment in this cell system. Right panel: Extracts of Jurkat cells untreated (lane 1) or treated with hydrogen peroxide for ten minutes (lanes 2-5) that were either pretreated with the MEK inhibitor PD98059 (lane 3), the JNK inhibitor SP600125 (lane 4), or the p38 inhibitor SB202190 (lane 5). Western blotting was performed as described above using the ETS1[pT38] antibody or an antibody recognizing total ETS1 protein. The data illustrate regulation of this phosphorylation site by the ERK branch of the MAPK signaling cascade. |
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 'BP7064 ETS1 (pT38)'.
