Project Grant R44GM148112

HIGH-THROUGHPUT WESTERN BLOTTING - PROJECT SUMMARY THE GOAL OF THIS PHASE II SBIR IS FOR BLOTTING INNOVATIONS TO COMMERCIALIZE A HIGH-THROUGHPUT, AFFORDABLE, AND ACCESSIBLE WESTERN BLOT. WESTERN BLOTTING, ONE OF THE MOST WIDELY USED PROTEIN ASSAYS IN BIOMEDICAL RESEARCH, ENABLES DETECTION OF SPECIFIC PROTEINS AND THEIR POST-TRANSLATIONAL MODIFICATIONS IN BLOOD, TISSUE, OR CELL LYSATE SAMPLES VIA ELECTROPHORESIS-BASED MOLECULAR WEIGHT SEPARATION. THE TECHNIQUE HAS REMAINED PRACTICALLY IDENTICAL TO WHEN IT WAS FIRST INTRODUCED IN THE LATE 1970S, AND IS OFTEN CONSIDERED A GOLD STANDARD FOR PROTEIN ANALYSIS. YET, WESTERN BLOTTING HAS BEEN REFRACTORY TO SCALE UP, TYPICALLY LIMITED TO ~10 SAMPLES PER RUN. CUSTOMER DISCOVERY INTERVIEWS SHOWED HIGH-THROUGHPUT WESTERN BLOTS RETAINING TRUSTED, FAMILIAR PROTOCOLS AND READOUTS, WITHOUT INCREASED CAPITAL AND OPERATING COSTS, WILL GENERATE DEMAND FROM ACADEMIC BIOLOGICAL SCIENCES LABS, PARTICULARLY PHARMACOLOGY AND SYSTEMS BIOLOGY. HOWEVER, THERE IS CURRENTLY NO AFFORDABLE, PRECISE, AND ACCESSIBLE HIGH THROUGHPUT SOLUTION AVAILABLE. TOTAL ADDRESSABLE MARKET IS ~$4 BILLION, SERVICEABLE ADDRESSABLE MARKET ~$1 BILLION, AND SERVICEABLE OBTAINABLE MARKET (SOM) ~$50 MILLION WITH ACADEMIC LAB BEACHHEAD. IN PHASE I, EXPERIMENTS AT BLOTTING INNOVATIONS AND AN INDEPENDENT ACADEMIC LAB ESTABLISHED THAT PRECAST 96-WELL POLYACRYLAMIDE GELS LOADED WITH MOLECULAR WEIGHT LADDER STANDARDS COULD BE RELIABLY SUBJECTED TO ELECTROPHORESIS IN OUR NOVEL HORIZONTAL APPARATUS, AND THEN TO STANDARD MEMBRANE TRANSFER. MOREOVER, WE WENT BEYOND THE ORIGINAL PHASE I AIMS AND TESTED THE SYSTEM WITH RECOMBINANT PROTEIN AND CELL LYSATES WHICH SHOWED BAND INTENSITY COEFFICIENT OF VARIATION (CV) ~5-20%, COMPARABLE TO EXISTING WESTERN BLOT METHODS. THUS, FEASIBILITY HAS BEEN ESTABLISHED. HERE, WE WILL COMMERCIALIZE TWO PRODUCTS: (I) A SUITE OF PRECAST 96-WELL GELS WITH GEL AND BUFFER CHEMISTRY OPTIONS AND (II) A NOVEL YET AFFORDABLE TANK FOR SUBMERGED HORIZONTAL ELECTROPHORESIS OF OUR GELS. AIM 1. EXPAND PROVEN APPLICATION BASE AND PRODUCT VARIATIONS. IN PHASE I WE FOCUSED ON FEASIBILITY WITH A SINGLE TYPE OF GEL, RUNNING BUFFER, TRANSFER PROTOCOL, AND LYSIS BUFFER. HOWEVER, THERE IS DEMAND FOR GREATER VARIETY. 1A. GEL AND ELECTROPHORESIS BUFFER CHEMISTRY. WE WILL CAST DIFFERENT % ACRYLAMIDE GELS WITH THE MAJOR CHEMISTRIES, AND TEST THEM WITH MOLECULAR WEIGHT LADDER AND MULTIPLE COMMON RUNNING BUFFERS. 1B. GEL SHELF-LIFE. WE WILL TEST SUITABLE GELS AS IN 1A AT 1, 3, 6 AND 12 MONTHS. 1C. LYSIS BUFFER. PHASE I USED ONLY RIPA / WHOLE CELL LYSATE WESTERN. WE WILL TEST MAIN VARIANTS WITH APPLICATIONS: NON-DENATURING, IMMUNOPRECIPITATION(IP)-WESTERN, AND NUCLEAR / CYTOPLASMIC FRACTIONATION. 1D. LOADING CONTROLS. PHASE I DID NOT ESTABLISH LOADING CONTROL PROTOCOLS. WE WILL TEST THE THREE MAIN APPROACHES: TOTAL PROTEIN STAINING, DUAL-COLOR FLUORESCENCE, AND STRIP/RE-PROBE WITH CHEMILUMINESCENCE. AIM 2. EVALUATE PRODUCT QUALITY FROM SCALED MANUFACTURING PROCESSES. TANKS ARE CURRENTLY 3D PRINTED AND PRECAST GELS ARE VERTICALLY CAST ONE-AT-A-TIME. WE HAVE DEVELOPED SCALABLE TANK (THERMOFORMING) AND GEL MANUFACTURING (HORIZONTAL) PROCESSES AND MADE INITIAL PRODUCTS. WE WILL VALIDATE SUITABLE QUALITY OF SUCH MANUFACTURED PRODUCTS BY APPLYING PHASE I EXPERIMENTS.