| Reversible protein phosphorylation plays a central role in numerous biochemical pathways and functions to alter protein activity and/or conformation (3,7). Methods for detecting protein phosphorylation have predominately relied upon the use of radioactive 32P for either the in vitro or in vivo phosphorylation reaction (2,6). To identify the specific amino acid residues that become phosphorylated on a specific protein, phosphoamino acid analysis is then required (2,3,6,7). The use of radioactivity and the multi-step analysis process which follows makes the entire process both hazardous and tedious. Over the past decade, antibodies specific for phosphotyrosine were developed, many of which can detect a single phosphorylated tyrosine residue (1,4,5). Although antibodies to phosphotyrosine have been relatively straight forward to produce, the extensive structural similarity between phosphoserine and phosphothreonine has contributed to the difficulty in raising highly specific antibodies to these phosphoamino acids (6). |
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| Catalog # | P4077-10 |
| Applications | Suitable for use in ELISA and Western Blot. Other applications not tested. |
| Recommended Dilution | ELISA: 0.1-1ug/ml |
| Western Blot: 1-5ug/ml |
| Optimal dilutions to be determined by the researcher. |
| Note | Milk-derived blocking solutions reportedly contain phosphoproteins that may inhibit phosphoamino acid antibody binding and therefore should be avoided. Membrane Blocking Solutions optimized for use with anti-phosphoamino acid antibodies provide enhanced blocking of non-specific signal. A 3% BSA solution may also be used. |
| Accessibility of the phosphothreonine residues within the native protein, and possibly the extent of protein phosphorylation, are likely to influence the effectiveness of this antibody. |
| Storage and Stability | May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. |
| Clone Type | Polyclonal |
| Isotype | IgG2a,k |
| Host | Mouse |
| Concentration | 0.5mg/ml |
| Form | Supplied as a liquid in PBS, pH 7.4, 0.1% sodium azide. |
| Purity | Purified by immunoaffinity chromatography. |
| Immunogen | Phosphothreonine containing proteins |
| Specificity | Recognizes Phosphothreonine. Reacts specifically with proteins containing phosphorylated threonine residues (phosphothreonine). Recognition of phosphothreonine containing proteins by this antibody appears to be independent of neighboring amino acids and species of origin of the phosphorylated protein. No significant crossreactivity with phosphoserine, phosphotyrosine either as free amino acids or in proteins has been observed. Has been confirmed to react with phosphothreonine containing proteins from lysates derived from EGF-stimulated A431 cells, HeLa cells, MCF-7 cells and Clone 9 (rat liver) cells. |
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| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
