Phosphorylation of proteins is a highly dynamic modification. Therefore, special care and attention needs to be applied when preparing samples for such analysis. You should also know that the ratios of phosphotyrosine:threonine:serine is 1:1700:3000 thus if you are interested mainly in tyrosine phosphorylation you should aim for pY enrichment (see below).
- Lysis should be conducted as quickly as possible. The lysis buffer depends on the experiment, please contact us for details.
- Cell lines, wash cells with cold PBS prior to lysis. We require at least 3e6 cells (preferable 6e6).
- Tissue, protein extraction must include physical grinding using bead-beating, homogenizer or mortar and pestle in the lysis buffer. We require at least 1mg extracted protein.
We use the Cell Signaling antibody, which requires at least 10mg lysate as starting point. We can detect approximately 600 pY sites from cells and approx. 200 sites from tissue.
We use the Cell Signaling anti K-e-GG antibody. The protocol requires at least 10mg extracted protein for optimal performance. Please contact us for further details.
We applied the OxiCAT method successfully for global analysis of redox-sensitive proteins.
We are currently working on several workflows for analysis of other post translational modifications. Please contact us for further information.