Journal
ANALYTICAL BIOCHEMISTRY
Volume 389, Issue 1, Pages 83-85Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.03.026
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The low specificity of anti-phosphoprotein antibodies is often a problem in immunoblotting analyses. We introduce simple pretreatment procedure for cell lysates to give more specific detection of phosphoproteins ill immunoblotting. Cellular phosphoproteins were preferentially trapped oil Phos-tag agarose phosphate-affinity beads in a homemade spin-centrifuge microtube unit, and nonphosphorylated proteins were excluded in the filtrate. The phosphoprotein-bound beads suspended in a sample-loading-dye solution were Subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting. We demonstrated improved detection of phosphorylated She and mitogen-activated protein kinase isoforms in A431 cell lysates by this new technique. (C) 2009 Elsevier Inc. All rights reserved.
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