Master of Science
Walter Henne, Jr., Ph.D.
Patty Fu-Giles, Ph.D.
Stephen Kent, M.B.A.
After purification of protein, it is important to know the concentration of protein in our samples. Concentration or amount of protein in the sample is determined by different assay as BCA (bicinchoninic acid) Assay, Bradford Assay, and Lowery Assay. There are different methods available to perform these assays, which have some limitations, restrictions, advantages and disadvantages. These methods require large amounts of costly reagents, proteins and most importantly, valuable time. To overcome these problems a new method is developed called Microdrop Protein Analysis. This new method required a very small amount of reagents and protein mixtures (2-5 µʅ volume) and is very quick and easy to perform. The main objective of this project was to develop either new or modified methods for detection and analysis of proteins using a newly introduced micro drop plate reader equipped with a 16 well microdrop reader (Take3 Plate). This Take3 Multi-Volume plate allows for easy and rapid analysis of 16 samples having as low as 2-µʅ volumes each by using Gen 5 software. The plate used in the instrument EpochTM micro plate spectrophotometer. We used commercially available BSA (bovine serum albumin) Protein as our test moiety. The concentration of protein in the solution was determined by BCA (bicinchoninic acids) Assay using UV-280 protocol or microdrop micro-BCA protocol provided by the manufacture (Pierce Chemical Company) with the appropriate controls (PBS blank and BSA test control protein). First, the experiment was carried out at room temperature to determine the lowest concentration of protein that we can use and get linear curve. Then, we did time-dependent studies at room temperature and heated the plate in a humidified incubator before measuring the absorbance. Then, we plotted a graph of concentration on the X-axis and absorbance on Y-axis to find out the concentration of protein. The overarching strategy is to develop robust assays that use minimum sample amounts given the high cost and limited 7 availability of many proteins. Successful completion of this work will benefit protein researches in rapid identification and analysis of protein during expression, analysis and isolation.
Khared, Hardik, "Determination of Protein Using Microdrop Assay" (2011). All Capstone Projects. 43.