UV Detection:  Simplest method. Place the protein in a quartz curette read the sample.   Almost all proteins have absorbence in the ultraviolet spectra.   What is actually being measured is the presence of phenylalanine, tyrosine and tryptophan.  The number of these in the protein is the determining factor in how strong the absorbence/mg. The more Phe Tyr and Trp the stronger the absorbence the fewer the weaker.  Proteins actually very widely in there absorbence but the rule of thumb is 1 AU= 1 mg/ml, The value for pure proteins  normally ranges from 0.4 to 2.4  although there are some proteins that have an absorbtivity of 0 AU/mg, no Phe Tyr, and Trp, up to as high as 20.  BSA a commonly used standard has an absorbtivity of 0.66AU/ for a 1 mg/ml solution.

The Folin- Lowry Method
One of the oldest amplified methods for proteins. There are a number of interfering reagents and the linked paper is a modified method to avoid some of the problems we will not be using the ptt step in the paper.   The basis of the method is the biuret color reaction which depends on the interaction of copper with the amide backbone of the protein. That reaction is then amplified with the Folin reagent which provides additional color.  The reaction is partially dependent on the amounts of Tyr and Trp in the protein and can vary significantly. The assay's range is from 20-400 µg/ml but is best used below 100 µg/ml.

The Bradford Method
This Method is a dye binding method using Coomassie Brilliant Blue G250, the G is important.  Bio-Rad makes a stabilized Bradford reagent which is commonly used and ignorant individuals may call this the Bio-Rad Method. Please correct them gently.  The Basis of the method is a chromatic shift in the dye when it binds to protein.  The dye interacts with both aromatic and positively charged residues. Variation in these residues will cause variation in the assay.   Detergents are a major interfering agent and the method is inappropriate when protein solutions contain those agents. this has a range from about 5-50 ug of protein in the assay

The BCA Method
Popularized by Pierce Biochemical this method is similar to the Folin-Lowry Method.  The Copper reagents are almost identical. It is somewhat more sensitive in the 5-100ug of protein in the assay.  The Pierce website is useful in understanding this assay.
 
 

The Lab
This task has you compare 4 methods for determining protein concentrations you will use Bovine Serum Albumin (BSA) Hexokinase and Lysozyme and create standard curves for each. Each of the methods has range of concentration where it works effectively.  You will need to design your protocol to reflect that.  

Please calculate dilutions of samples appropriately. You will need to generate sample curves for all methods starting with 2 mg/ml protein (This is a standard protein concentration that is regularly found in labs.).  BCA is a Kit that you will use. You will need to make the Lowry Assay reagents The Bradford reagent and the copper reagent for the BCA assay.  The Bradford reagent takes over night to go into solution so you will need to come in on the following day to filter your reagent.   .
The methods that we will be comparing will be the

Lowry assay
Bradford assay
BCA assay
280 absorbance
 
Week One  make stock reagents
Week Two perform Assays

Some questions to think about.  Feel free to address others.
Why are there upper limits on the concentrations for the assay?
Do the different assays give different responses for the samples?
What method would you choose for a protein that requires detergent in its storage/purification buffer?
Which would you choose to follow protein concentration during purification?