Anti-Phosphotyrosine antibody produced in rabbit
SIGMA/T1325 - 0.5 mg/mL, affinity isolated antibody, buffered aqueous solution
Synonym: Anti-p-Tyr
MDL Number: MFCD00164715
Product Type: Chemical
Immunoblotting
Whole cell extracts of A431cells (Lane A) untreated or (Lanes B-H) EGF-treated (1 μg/mL human EGF, Cat. No. E9644 for 5 min) were separated on SDS-PAGE, blotted with Rabbit Anti-Phosphotyrosine (Cat. No. T1325) and developed using Goat Anti-Rabbit IgG-Peroxidase (Cat. No. A0545) and a chemiluminescent substrate.(Lane A) serum-starved A431 cells; (Lane B) EGF treated A431 cells. Antibody concentration 0.3 μg/mL; (Lane C) EGF treated A431 cells. Antibody concentration 0.15 μg/mL. (Lanes D–G) EGF treated A431 cells. Antibody concentration 0.3 μg/mL. Inhibition with antigen (20 μg/mL): (Lane D) Inhibition with phospho-L-tyrosine (inhibition), (Lane E) Inhibition with L-tyrosine (no inhibition), (Lane F) Inhibition with phospho-L-serine (no inhibition), (Lane G) Inhibition with phospho-L-threonine (no inhibition); (Lane H) EGF treated A431 cells. No primary antibody added (negative control)
Whole cell extracts of A431cells (Lane A) untreated or (Lanes B-H) EGF-treated (1 μg/mL human EGF, Cat. No. E9644 for 5 min) were separated on SDS-PAGE, blotted with Rabbit Anti-Phosphotyrosine (Cat. No. T1325) and developed using Goat Anti-Rabbit IgG-Peroxidase (Cat. No. A0545) and a chemiluminescent substrate.(Lane A) serum-starved A431 cells; (Lane B) EGF treated A431 cells. Antibody concentration 0.3 μg/mL; (Lane C) EGF treated A431 cells. Antibody concentration 0.15 μg/mL. (Lanes D–G) EGF treated A431 cells. Antibody concentration 0.3 μg/mL. Inhibition with antigen (20 μg/mL): (Lane D) Inhibition with phospho-L-tyrosine (inhibition), (Lane E) Inhibition with L-tyrosine (no inhibition), (Lane F) Inhibition with phospho-L-serine (no inhibition), (Lane G) Inhibition with phospho-L-threonine (no inhibition); (Lane H) EGF treated A431 cells. No primary antibody added (negative control)
Immunoblotting
Whole cell extract of EGF-treated A431cells was separated on SDS-PAGE and blotted with Rabbit Anti-Phosphotyrosine antibodies:
Lanes
A. Anti-Phosphotyrosine, (Cat. No. T1325) 0.3 μg/mL
B. Anti-Phosphotyrosine, (pY-256), (Cat. No. T1575) 0.3 μg/mL
C. Anti-Phosphotyrosine (PY-20), (Cat. No. P4110) 0.3 μg/mL
D. Anti-Phosphotyrosine (PT154), (Cat. No. P1869) 0.3 μg/mL
The antibodies (Lanes A,B) were developed with Goat Anti-Rabbit IgG-Peroxidase conjugate (Cat. No. A0545) and a chemiluminescent substrate. The monoclonal antibodies (Lanes C,D) were developed with Goat Anti-Mouse IgG-Peroxidase conjugate (Cat. No. A2304) and a chemiluminescent substrate.
Whole cell extract of EGF-treated A431cells was separated on SDS-PAGE and blotted with Rabbit Anti-Phosphotyrosine antibodies:
Lanes
A. Anti-Phosphotyrosine, (Cat. No. T1325) 0.3 μg/mL
B. Anti-Phosphotyrosine, (pY-256), (Cat. No. T1575) 0.3 μg/mL
C. Anti-Phosphotyrosine (PY-20), (Cat. No. P4110) 0.3 μg/mL
D. Anti-Phosphotyrosine (PT154), (Cat. No. P1869) 0.3 μg/mL
The antibodies (Lanes A,B) were developed with Goat Anti-Rabbit IgG-Peroxidase conjugate (Cat. No. A0545) and a chemiluminescent substrate. The monoclonal antibodies (Lanes C,D) were developed with Goat Anti-Mouse IgG-Peroxidase conjugate (Cat. No. A2304) and a chemiluminescent substrate.
Immunofluorescence
Plasma membrane and cytoplasmic staining: A431cells fixed with paraformaldheyde were stained with Anti-Phosphotyrosine (Cat. No. T1325) at 3 μg/mL (A and C) and 1.5 μg/mL (B) followed by Goat Anti-Rabbit IgG-FITC (Cat. No. F9887). Panels A, B - Serum starved A431 cells stimulated with human EGF (1 μg/mL, 5min., EGF human, Cat. No. E9644). Panel C - Untreated control A431cells.
Plasma membrane and cytoplasmic staining: A431cells fixed with paraformaldheyde were stained with Anti-Phosphotyrosine (Cat. No. T1325) at 3 μg/mL (A and C) and 1.5 μg/mL (B) followed by Goat Anti-Rabbit IgG-FITC (Cat. No. F9887). Panels A, B - Serum starved A431 cells stimulated with human EGF (1 μg/mL, 5min., EGF human, Cat. No. E9644). Panel C - Untreated control A431cells.
| antibody form | affinity isolated antibody |
| antibody product type | primary antibodies |
| biological source | rabbit |
| clone | polyclonal |
| concentration | 0.5 mg/mL |
| conjugate | unconjugated |
| form | buffered aqueous solution |
| Quality Level | 200  |
| shipped in | dry ice |
| storage temp. | −20°C |
| technique(s) | indirect immunofluorescence: 3-6 μg/mL using A431 stimulated by human EGF. |
| western blot: 0.3-0.6 μg/mL using total cell extract of A431 stimulated by human EGF |
| Application: | Anti-Phosphotyrosine antibody is suitable for use in indirect immunofluorescence (3-6 μg/mL using A431 stiμLated by human EGF) and immunoblot (using U2OS Dr1 cells ). The antibody may also be used for immunoprecipitation (1:40 using rat lumbar and thoracic lysates) and immunocytochemistry. |
| Biochem/physiol Actions: | Protein phosphorylation is the most abundant among post-translational modifications of cellular proteins, regulating intracellular signal transduction pathways in every living cell. Tyrosine, serine and threonine are the major amino acids that are phosphorylated in proteins. Antibodies specific for phosphotyrosine are essential tools for the characterization of tyrosine phosphorylation in many signal transduction pathways. |
| Disclaimer: | Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. |
| Immunogen: | phospho-L-tyrosine conjugated to KLH |
| Physical form: | Solution in 0.01 M phosphate buffered saline, pH 7.4, and 1% BSA, containing 15 mM sodium azide. |
| Specificity: | Anti-Phosphotyrosine recognizes tyrosine phosphorylated proteins. |
| Storage and Stability: | For extended storage, freeze in working aliquots. Repeated freezing and thawing is not recommended. Storage in "frost-free" freezers is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use. Working dilution samples should be discarded if not used within 12 hours. |
| RIDADR | NONH for all modes of transport |
| WGK Germany | nwg |
| Flash Point(F) | Not applicable |
| Flash Point(C) | Not applicable |
| Storage Temp. | −20°C |
| UNSPSC | 12352203 |