Hello!
I want to quantify by ELISA the secreted (from platelets poor plasma) and the non-secreted (from platelet lysate) PF4 before and after TRAP stimulation.
I will use the ELISA from R&D #DY795.
I have found different buffers to lyse plateles (e.g., TNE,Triton alone, RIPA [with and without SDS] ) but I am still not sure which one would disrupt the membranes of the platelet and the granules.
I have been trying to fine a good reference about the membrane of the granules but I cant find any. So my questions are:
1- Have anyone tried to lyse platelets using different buffers and compare the results?
2- are the membranes of the platelets the same as the granules? (i.e., if a buffer can lyse the cell wall would it have the same effect in granules?)
3-. Have anyone worked with this ELISA or know if it detects denatures PF4 in case I choose a buffer that might denature my protein?
4. Related question... Can PF4 be phosphorylated or cleaved so it justify using protease and/or phosphatase inhibitors?
Thanks in advance for your answers!!
you might try passing the platelets through decreasing bore size (diameter) needles. Mechanical stimulation is the old man's trick to degranulate platelets. ADP sounds like a great, more physiological stimulator.
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