When analyzing cell fractions it is important to determine the concentration of protein in a particular sample. There are several chemical assay systems used to detect protein, including the Lowry assay and the BCA or bicinchoninic acid assay. BCA in the form of its water-soluble salt, is a sensitive, stable and highly specific reagent for the cuprous ion (Cu+). Peptide bonds and four amino acids (cysteine, cystine, tryptophan, and tyrosine) have been reported to be responsible for color formation in protein samples when assayed with BCA.
The reaction scheme below shows how the BCA protein assay combines the biuret reaction (protein reducing Cu++ in an alkaline medium to produce Cu+) with the unique features of BCA. The purple reaction product, formed by the interaction of 2 BCA molecules with one cuprous ion (Cu+), is water soluble and exhibits a strong absorbance at 562 nm. This allows the spetrophotometric quantitation of protein in aqueous solutions.
We will be performing the BCA assay on our cell fractions from the last lab exercise. Dilutions will be made of the fractions and a series of dilutions of a standard protein (bovine serum albumin or BSA) solution. Each dilution will then be assayed with the BCA reagents. From the BSA standard dilutions you will obtain a standard curve for absorbance related to protein concentration. This will then be utilized to determine the concentration in dilutions of your cell fractions. This information will be necessary for the next lab exercise, in which we will assay for marker enzymes based on the protein content.
1. Make a series of 10 fold dilutions of your sample fraction in TE buffer--3 dilution tubes plus the original full strength will be assayed. You only need 200ul of each dilution. PLEASE CALCULATE THESE DILUTIONS BEFORE COMING TO CLASS!!
2. Make the following dilutions in TE of the standard BSA solution (2 ug/ul) in 200ul total volume (note only some groups will do the standard--I'll assign who does what.)
AGAIN PLEASE CALCULATE THESE BEFORE COMING TO CLASS!!
Amount of BSA (ug) ul BSA ul lysis buffer total ul Dilution
3. Get clean assay tubes--2 tubes for every dilution you will assay--and label them. Duplicates or triplicates are done to allow for experimental error.
4. The BCA reagent (prepared) consists of 50 parts reagent A and 1 part reagent B.
5. Into each assay tube place 2 ml of BCA reagent. Then put 0.1 ml (100 ul) of the protein dilution (fraction or standard) to be assayed into the appropriate tubes. Mix well, then incubate at 37oC for 30 minutes.
6. Cool to room temperature and measure the absorbance of each sample at 562 nm with a spectrophotometer. The use of this will be demonstrated.
7. Graph your standard values, on regular graph paper, draw your standard line and use it to determine the concentration of protein in your cell fraction dilutions.