How To Setup Microplates for Protein and DNA Quantitative Assays
This tutorial demonstrates a quick and easy technique for setting up microplates for BCA, Bradford, Lowry and Fluorescence DNA assays. It is fast to pipette, takes full advantage of the dynamic range of these assays, and works seamlessly with the BioTek's Quantitative Assay Apps. This method will help get the read step quickly, especially for those who have traditionally used a cuvette-based measurement or have relied on 1.5mL tubes for setting up samples.
Hello. My name is Joseph Negri and I'm a Product Manager here at BioTek Instruments. While completing my PhD in neuroscience, I regularly had to perform BCA assays in order to quantify the protein content of my experimental samples. For many of us in life science labs protein quantification assays such as BCA, Bradford and Lowry are a regular part of our day to day work. For this reason, the team here BioTek has designed our new Quantitative Assays App to help make these routine lab tasks a bit faster and easier.
Before demonstrating the Quantitative Assays App I want to show you a technique that I regularly use in the lab for setting up these types of assays in microplates. This method should help you get to the read step quickly especially for those of you who have traditionally used cuvette-based measurement or have relied on 1.5 mil tubes for setting up your samples. This approach is fast to pipette, takes full advantage of the dynamic range of these assays, and works seamlessly with the Quantitative Assays App downstream.
For demonstration purposes I'm going to run a BCA assay. Using this method, I'm going to use one microplate to set up my BCA in a second microplate. In the first plate I'm going to make up my standard curve and create my sample dilutions. I will create a 7-point standard curve ranging from 2000 micrograms per mil to 175 micrograms per mil by performing a 1.5-fold serial dilution. For BCA assay I know that I will need 25 microliters of standard or sample for each well, so I want to have 100 microliters of each solution in order to account for dead volume.
To do this I'll use a single column of a 96 well plate. In the first well I'll add 300 microliters of the 2000 microgram per mil BSA standard provided with the assay kit. In the next seven wells, I'll add 100 microliters of water. I will take 200 microliters and mix it with 100 microliters in the subsequent well to perform the 1.5-fold dilution. When I aspirate from the seventh well, I'll discard the tip leaving the eighth well as water alone to serve as my blank. The 7-point standard curve effectively covers the detection range of the BCA assay. I do not need to remember the intervening concentrations since these will be readily calculated using the Quantitative Assays App as I will demonstrate in a subsequent video.
Having setup my standards I'll use a similar technique on the right-hand side of the plate and generate my sample dilutions. In this case I'll perform a twofold dilution in order to cover a wider concentration range in fewer steps. Starting with 200 microliters of each experimental sample I will dilute 100 microliters of each, one to two, one to four, and one to eight in each subsequent row. Now taking my second plate, I will lay out my plate map. I'll designate the area in which I will include my seven standards, and my blank. I'll also designate the area in which I plan to include my sample replicates. I have samples one, two, three, and four. And I have dilutions 1, 1 to 2, 1 to 4, and 1 to 8.
Taking the first plate, I'll turn my replicates of each sample to the second plate in which the BCA reagent will be added. I'll now add the BCA reagent to the plate and incubate at 37 degrees for thirty minutes. At this point I can use the Quantitative Assays App to set my plate map, fit the standard curve and normalize my samples.