This question is posted on behalf of Patricia L. Yeyati, please reply to the mailing list and not directly to me. /Maria ------------------------------------------------------------------------ I am trying to do dual staining of a transcription factor and DNA by paraformaldehide fixation and PI for DNA content. I had excellent results with Schmidth methods (0.25% PFA and Tween permeabilization)when I over-express my protein in a myeloid cell line, but when I try to do the same with embryonic stem cells (which endogenously express my proitein) I either get good protein staining or DNA but never in the same sample so I can never identify the cell cycle stage of cells that express my transcription factor. I have tryed with ethaniol fixation but I looose protein staining and even with PFA fixation I loose protein detection in about 30% when I add PI, which then makes positive cells look almost the same as the controls. I am afraid that becouse my experimental protein is bound to DNA thehen PI intercalates with the DNA displaces this protein and that is its detection is so decraesed. 1) should I change fixation conditions? 2) Why does PI prescence decrease protein detection? Any sugestion is welcomed. Thank you in advance. Patricia -- Patricia L. Yeyati, PhD John Hughes Bennett Laboratory - Leukaemia Research Fund University of Edinburgh Western General Hospital Tel: +44-131- 537-- 3155 Crewe Road Fax: +44-131- 537 3160 Edinburgh EH4 2XU http://www.ed.ac.uk/ UK
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