BSA Protein Standard Curve - Biuret Assay

welcome to my lab i'm drew colump today in the lab we're going to be doing a protein standard curve we're going to use a biuret test as it binds to the proteins or peptides and changes color as you can see the blank shows the biored itself is blue without any proteins or peptides

in solution as we add more protein you can see the color changes to a more purple color in this lab i will demonstrate adding protein to the biuret reagent and you can see the colorimetric reaction we will then use a genesis 30 spectrophotometer and we will use the highest concentration bsa we created which is 10

milligrams per ml we will create an absorption spectrum for this the protein in the biuret test and we will determine what the a max is for this particular solution using this maximum absorbance we will then perform a standard curve we've done a number of dilutions we will read off the percent transmittance from the spectrophotometer

we will do a conversion from percent transmittance to absorbance using a equals 2 minus the log percent transmittance we will then use this data to create a standard curve graph with concentration bsa along the horizontal axis and absorbance along the vertical axis we will plot our data points on this graph connecting it with a nice straight

line following that a colleague of mine prepared two unknown samples i will then attempt to determine what the concentration is i was told it is below 10 milligrams per mil let's begin by observing what happens when we add protein to the bioret reagent here we have protein in this micro pipet we add it to the bioret reagent mixed

thoroughly you can see it changes from a blue color to a purple color the standards we will use today are bsa bovine serum albumin it is a protein found in calcium we have diluted down samples from 10 milligrams per ml all the way down to 0.625 milligrams per mil as our standards the blank we just added distilled water