specific ser/Thr phosphorylation detection by in house antibody - (Jan/04/2007 )
Hi:
I am having trouble to detect specific Thr phosphorylation by our in-house Thr phosphorylation antibody. By bioinformative softwares and also in agreement with other labs, we identify one Thr phosphorylation site located in putative PKC activation site. Following, we generate this specific Thr residue antibody and affinity purify this antibody by phosphopeptide. when i did western blot by either using activator (PMA in this case) or mimcking phosphorylation (mutagenesis), i could not pick up any signals. So any experience, suggestions or tips from your guys
-yxz98-
QUOTE (yxz98 @ Jan 5 2007, 08:09 AM)
Hi:
I am having trouble to detect specific Thr phosphorylation by our in-house Thr phosphorylation antibody. By bioinformative softwares and also in agreement with other labs, we identify one Thr phosphorylation site located in putative PKC activation site. Following, we generate this specific Thr residue antibody and affinity purify this antibody by phosphopeptide. when i did western blot by either using activator (PMA in this case) or mimcking phosphorylation (mutagenesis), i could not pick up any signals. So any experience, suggestions or tips from your guys
I am having trouble to detect specific Thr phosphorylation by our in-house Thr phosphorylation antibody. By bioinformative softwares and also in agreement with other labs, we identify one Thr phosphorylation site located in putative PKC activation site. Following, we generate this specific Thr residue antibody and affinity purify this antibody by phosphopeptide. when i did western blot by either using activator (PMA in this case) or mimcking phosphorylation (mutagenesis), i could not pick up any signals. So any experience, suggestions or tips from your guys
one should try to generate more than one clone (for mAb) or serum (for pAb), only one serum may not yieled in an effective binding antibody; nevertheless, one should try to optimum binding conditions; try different pH and salt conc; try PBS and TBS for improvement of Wb
are you sure that the Thr of the binding site is an cis-phosphorylation site?; or is it rather trans-phosphorylated by PDK-1?
-The Bearer-