Project Grant R41GM148112

ACCESSIBLE AND ROBUST HIGH-THROUGHPUT WESTERN BLOTTING FOR SMALL SAMPLE SIZES - PROJECT SUMMARY THE GOAL OF THIS PHASE I STTR IS FOR BLOTTING INNOVATIONS, LLC TO ESTABLISH FEASIBILITY OF A COMMERCIALIZABLE MESOWESTERN-A HIGH-THROUGHPUT, AFFORDABLE WESTERN BLOTTING TECHNIQUE THAT WE RECENTLY DEVELOPED. WESTERN BLOTTING IS A TECHNIQUE FOR MOLECULAR-WEIGHT-RESOLVED ANALYSIS OF PROTEINS AND THEIR POST-TRANSLATIONAL MODIFICATIONS THAT IS PRACTICED TODAY ALMOST IDENTICALLY TO WHEN IT WAS FIRST INTRODUCED IN THE LATE 1970S. IT REMAINS ONE OF THE MOST WIDELY-USED PROTEIN ASSAYS ACROSS BIOMEDICAL RESEARCH, PERHAPS THE MOST USED IN THE PAST 10 YEARS. MAJOR REASONS ARE THAT IT IS LOW-COST, OFTEN A GOLD STANDARD, AND WELL-ESTABLISHED IN MOST LABS. YET, WESTERN BLOTTING HAS BEEN REFRACTORY TO SCALE UP, TYPICALLY LIMITED TO ~10 SAMPLES PER RUN. CAPILLARY ELECTROPHORESIS-BASED SEPARATION IN AUTOMATED APPARATI HAS BEEN DEVELOPED THAT INCREASE THROUGHPUT WITH SMALLER SAMPLES, BUT ARE EXPENSIVE AND CAN BE SENSITIVE TO SAMPLE PREPARATION. THE MICROWESTERN USES PIEZOELECTRIC PIPETTING FOR UP TO 96 BLOTS AT A TIME IN A STANDARD FOOTPRINT; HOWEVER, THE PIEZOELECTRIC APPARATUS IMPOSES CAPITAL COST AND TECHNICAL DIFFICULTLY DETERRENTS. WE ESTABLISHED THE MESOWESTERN THAT ANALYZES OVER 300 SAMPLES WITH A SIMILAR FOOTPRINT, AFFORDABILITY, AND EASE-OF-USE AS TRADITIONAL WESTERN BLOTS, AND WITH ~10-FOLD LOWER SAMPLE SIZE REQUIREMENTS. OUR MAIN PRODUCTS ARE A PRECAST MESOWESTERN GEL THAT IS LOADABLE BY A LOW-COST PIPETTING ROBOT (OPENTRONS) AND A NOVEL YET AFFORDABLE TANK FOR IMMERSED HORIZONTAL ELECTROPHORESIS OF THE LOADED PRECAST GEL. A MAIN INNOVATION IS A CUSTOMIZABLE GEL CASTING DEVICE THAT PRODUCES POLYACRYLAMIDE GELS WITH HUNDREDS OF ~1 UL WELLS, AND ASSOCIATED PROTOCOLS FOR ROBUST GEL CASTING AND ELECTROPHORESIS. ANOTHER MAIN INNOVATION IS IMMERSED HORIZONTAL TANK ELECTROPHORESIS FOR POLYACRYLIMIDE GELS; ONLY SEMI-DRY HORIZONTAL (MICROWESTERN) OR IMMERSED VERTICAL TANK (TRADITIONAL) ARE CURRENTLY AVAILABLE. PHASE I HYPOTHESIS. CAN PRECAST MESOWESTERN GELS BE ROBUSTLY-LOADED ROBOTICALLY, AND THEN SUBJECTED TO IMMERSED HORIZONTAL TANK ELECTROPHORESIS? WE HYPOTHESIZE THAT THIS CAN BE ACCOMPLISHED BY DESIGNING A RIGID INSERT THAT HOLDS THE GEL DURING CASTING AND SHIPPING BUT ALSO FITS INTO (I) OPENTRONS PIPETTING ROBOTS AND (II) A LOW-COST, HORIZONTAL IMMERSED ELECTROPHORESIS APPARATUS. WE HAVE TWO AIMS THAT WILL ESTABLISH FEASIBILITY OF THE PRODUCT BY TESTING THIS HYPOTHESIS. IN AIM 1, WE WILL ESTABLISH ROBUST ROBOTIC LOADING OF SHIPPED, PRECAST MESOWESTERN GELS. WE FOCUS ON OPENTRONS MICROPIPETTING ROBOTS THAT ARE EASY TO USE AND VERY AFFORDABLE. IN AIM 2, WE WILL ESTABLISH ROBUST ELECTROPHORESIS OF ROBOTICALLY-LOADED MESOWESTERN GELS. SUCCESS IN EACH AIM IS DEFINED BY VARIABILITY (CV%) ACROSS ANALYTES AND TECHNICIANS TO BE