BIO273 – Experiment 1 – Absorption Spectrum & Standard Curve for Biuret Assay of BSA

BIO273 – Experiment 1 – Absorption Spectrum & Standard Curve for Biuret Assay of BSA.

Step 1: Start with an 8 mg/mL Bovine Serum Albumin (BSA) solution, prepare a series of 5 standards that range in concentration from 0-8 mg/mL (do not include 0 or 8 mg/mL as any of your standards).

Fill in the table on the assignment sheet and briefly summarize below how you will prepare each standard. An example is given.

The total volume of each dilution will be 4 mL. You will make them directly into the cuvettes that go into the spectrophotometer. Note the diluent is a phosphate-buffered saline (PBS) solution, not water.

*In this example, I want to make a 2 mg/mL dilution. 2 mg/mL is ¼ of the working stock concentration of 8 mg/mL (I did 2 divided by 8 to get 1/4).

If I am doing a 1/4 dilution and making 4 mL total, then 4 x 1/4 = 1 mL. I need to pipette 1 mL of the 8 mg/mL solution into the test tube. To make 4 mL total, I need to add 3 mL of water to this. (Subtract 4 mL total - 1 mL stock = 3 mL). 

Repeat this for each of the concentrations you choose.  The higher the concentration, the more stock you will use. If your values are too weird - like need 3.666666 mL, then choose a different concentration or dilution factor, since this is not practical and will lead to error. Try to keep the volumes to whole numbers or decimals that do not repeat. 

Please note the concentration unit throughout this entire experiment is always mg/mL (milligrams per millilitre). You do not need to do any unit conversions. We are always keeping this same unit for concentration.

Summarize how you will prepare each of the standards below:

Example:

Standard 1: Concentration ____2 mg/mL______

Pipette 1 mL using 1000 µL micropipette into a cuvette labelled “2”.

Pipette (new tip) 3 mL using 1000 µL micropipe into same cuvette, mix with that tip.

Assignment Sheet – Standard Curve of Biuret Protein Assay for Quantification of BSA

1. Preparation of Standards [4 marks]

Summary:            [6 marks]

Standard 1: Concentration _____________

How to prepare:

Standard 2: Concentration _____________

How to prepare:

Standard 3: Concentration _____________

How to prepare:

Standard 4: Concentration _____________

How to prepare:

Standard 5: Concentration _____________

How to prepare:

2. Suppose you collect the following data for the given BSA Standards.

a) Fill in the absorbance column using the %T data. (Show one calc on the side) [2]

b) Create a standard curve graph, by hand (title, label axes (include units) and draw line of best fit)           [5]

c) Determine the concentration of the unknown from the given data and complete the table.                                  [3]

Please answer page 2 and 3 only of the attached document