User:Michael S. Bible/Notebook/571/2014/09/10
Biomaterials Design Lab | Main project page Previous entry Next entry |
Bradford AssayProcedure taken from Dr. Fox's page DescriptionThe most direct method for measuring the protein concentration is the use of the Beer - Lambert Law, using published extinction coefficients (molar absorptivities) for the proteins at λ = 280 nm in a UV-VIS spectrum. For low concentrations of proteins, UV-VIS of just the protein is often not sensitive enough to accurately measure concentration. (The limit of detection is about 2 - 3 μM for most proteins.) During the semester, we will likely need to measure protein concentrations that are lower than this. In addition, molar masses and/or extinction coefficients of some proteins are not well quantified. One tool we have can use to measure protein concentrations on the μg/mL level is called the Bradford Assay. The Bradford Assay makes use of the Coomassie Blue dye, which binds to proteins. Upon binding to a protein, this dye undergoes a change in its absorption features. (No protein: peak at 460. Protein: peak at around 600). We will be making calibration curves (using the Bradford Assay) for the different proteins we'll be using throughout the semester. Since this method depends on the number of peptide bonds, concentrations are reported by mass and the method is fairly independent of the particular protein being measured. There are a few interferences, such as co-factors that absorb near λ = 600 nm (e.g. hemes) or basic pH buffers.
TasklistThe basic protocol can be found here (*Note: use section 2.3, page 5) or here.
DataNotesThis area is for any observations or conclusions that you would like to note.
|