Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates.
Activation of various cell types by different agonists is known to stimulate a transient increase in the level of tyrosine phosphorylation of certain cellular proteins. Such phosphorylation is essential for mediating signalling by these agonists. The preservation of the tyrosine phosphorylation of proteins in lysates has proven to be a difficult task in neutrophils because of their large arsenal of proteases and phosphatases. Here we describe a technique that we found useful for preserving the tyrosine phosphorylation of cellular proteins. The technique depends on the denaturing lysis of neutrophils followed by the removal of the denaturing agents using Sephadex columns. Preparing neutrophil lysates by this technique has proven to be reliable in terms of maintaining the stability of the tyrosine phosphorylated proteins of various molecular weights and their subsequent immunoprecipitation and identification.
al-Shami
A
A
Centre de Recherche en Rhumatologie et Immunologie, Laval University, Ste Foy, Québec, Canada.
Gilbert
C
C
Barabé
F
F
Gaudry
M
M
Naccache
P H
PH
D000328:Adult; D002458:Cell Fractionation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006358:Hot Temperature; D006801:Humans; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry*; D019000:Phosphotyrosine / Q000378:metabolism*; D011489:Protein Denaturation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases; D019061:src-Family Kinases / Q000378:metabolism
D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C067419:CBL protein, human