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Synthetic phosphopeptide derived from human SP1 around the phosphorylation site of threonine 453 (I-R-TP-P-T).
Our Abpromise guarantee covers the use of ab59257 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration.|
|WB||1/500 - 1/1000. Detects a band of approximately 90 kDa (predicted molecular weight: 81 kDa).|
|ChIP||Use at an assay dependent concentration. PubMed: 20375015|
|ICC/IF||Use at an assay dependent concentration. PubMed: 21115498|
Immunofluorescence analysis of HeLa cells, using ab59257 Antibody. The picture on the right is treated with the synthesized peptide.
Wild type MEF cells (2 × 107 cells) were cross-linked with formaldehyde, quenched with glycine, resuspended in SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.0, with protease inhibitors and phosphatase inhibitors), sonicated on ice, and centrifuged at 4 °C. Supernatant (400 μl) were diluted to a final volume of 4 ml in a mixture of 9 parts dilution buffer (1% Triton X-100, 150 mm NaCl, 2 mm EDTA, 20 mm Tris-HCl, with protease inhibitors, pH 8.0) and 1 part lysis buffer.
Mixtures were incubated with 4 μg of anti-p-Sp1 (ab 59257) or anti-Sp1 antibodies with rotating at 4 °C overnight followed with incubation with 100 μl of protein A beads with rotating at 4 °C for 4 h. After gentle centrifugation (2000 rpm), beads were resuspended in 1 ml of wash buffer (1% Triton X-100, 0.1% SDS, 150 mm NaCl, 2 mm EDTA, 20 mm Tris-HCl, with protease inhibitors, pH 8.0) and washed with wash buffer 3 times followed by one wash with a final wash buffer (1% Triton X-100, 0.1% SDS, 500 mm NaCl, 2 mm EDTA, 20 mm Tris-HCl, pH 8.0, with protease inhibitors). The immune complexes were eluted with elution buffer (1% SDS, 100 mm NaHCO3) followed by incubation with proteinase K and RNase A (500 μg/ml each) at 37 °C for 30 min. Reverse cross-links were performed by placing the tubes at 65 °C overnight. Immunoprecipitated DNA was extracted and dissolved in sterile water and Q-PCR was performed.
ab59257 (1:1000) Antibody detects endogenous levels of SP1 only when phosphorylated at Thr453.