Development of a fluorescent F-actin blot overlay assay for detection of F-actin binding proteins.
Interactions between cellular proteins and filamentous (F) actin are key to many cellular functions, e.g., cell motility, endocytosis, cell:cell adhesion, and cell:substrate adhesion. Previously, a functional assay using 125I-labeled F-actin to detect a subset of F-actin binding proteins by blot overlay was developed. We have modified this assay to use the fluorescent label, Alexa 488, in place of 125Iodine. The detection limit for Alexa 488-labeled actin using a Molecular Dynamics STORM 860 Fluorescence/PhosphorImager was as little as 100pg of labeled actin. The Alexa 488 F-actin assay detects the same proteins from Dictyostelium discoideum and with approximately the same sensitivity (approximately 10 microg/ml F-actin final concentration) as the analogous 125I-labeled F-actin blot overlay. The use of Alexa 488 F-actin for blot overlay assays requires no radioactive materials and generates no hazardous waste. Assays can be performed on the laboratory bench top and the blots imaged directly with a blue laser scanner, either wet or dry. In addition, the Alexa 488 fluorophore is highly resistant to photobleaching, does not decay, and may be stored frozen or lyophilized. Alexa 488 F-actin is a stable, cost-effective, nonhazardous probe used for rapid identification of a subset of F-actin binding proteins.
Hitt
Anne L
AL
Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA. hitt@oakland.edu
Laing
Suzette D
SD
Olson
Stacey
S
D000199:Actins / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western / Q000379:methods*; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D004023:Dictyostelium / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry*; D008840:Microfilament Proteins / Q000032:analysis* / Q000378:metabolism; D011817:Rabbits; D012680:Sensitivity and Specificity; D012965:Sodium Chloride / Q000494:pharmacology
D000199:Actins; C417561:Alexa 488 hydrazide; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D005456:Fluorescent Dyes; D006834:Hydrazines; D008840:Microfilament Proteins; D012965:Sodium Chloride