Western blotting as a method for studying cell-biomaterial interactions: the role of protein collection.
Research of cell-biomaterial interactions is building on knowledge and methods available in cell and molecular biology. Western blotting is one of the options to characterize protein expression in cell populations. Method transfer to biomaterial model systems is not trivial because of the structure that exists in many biomaterials, preventing the collection of cell lysate by mechanical means. In this technical report, we describe the influence of different protein collection methods in a model system for cell-biomaterial interactions, consisting of endothelial cells exposed to different stimuli. In particular, the influence of trypsinization before lysis, and handling complexity were determined. The results of this study indicate that many changes in proteins occur because of the intermediate enzymatic treatment, despite the use of ice-cold solutions and protease and tyrosine phosphatase inhibitors throughout the procedure. Protein degradation and slight depressions in molecular weight were observed. The enzymatic treatment induced a changed cell status associated with detachment from the substratum. Western blotting of lysates of cells obtained through enzymatic harvest therefore can only be used with internal controls for the assessment of artifacts introduced by trypsinization, or alternative methods should be sought.
van Kooten
T G
TG
Institute of Pathology, Johannes Gutenberg University, Mainz, Germany. t.g.van.kooten@med.rug.nl
Klein
C L
CL
Kirkpatrick
C J
CJ
D015703:Antigens, CD; D001391:Azo Compounds; D001672:Biocompatible Materials / Q000633:toxicity*; D015153:Blotting, Western / Q000379:methods*; D015820:Cadherins / Q000378:metabolism; D002478:Cells, Cultured; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008422:Materials Testing / Q000379:methods*; D019000:Phosphotyrosine / Q000096:biosynthesis / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D000099152:Cadherin 5
D015703:Antigens, CD; D001391:Azo Compounds; D001672:Biocompatible Materials; D015820:Cadherins; D004396:Coloring Agents; D019000:Phosphotyrosine; D011506:Proteins; D016159:Tumor Suppressor Protein p53; D000099152:Cadherin 5; C032756:ponceau S