I know how to carry out an absorption spectrum I just need help on how to to prepare my test tubes and what to put in then. I am very confused and any help would be greatly appreciated. THANKS !
You will be given a sample containing an unknown concentration of protein (Bovine Serum Albumin). The unknown concentration of the bovine serum albumin will be between 0 and 10 mg/mL in 1% (w/v) NaCl. You are to determine the protein concentration in the sample, using the Biuret assay as described below.
Do all of your planning and make your calculations before you come to class. You will not be permitted to bring any notes into the exam. You are not permitted to ask any of the college professors for help with the preparations for this exam. You may, however, ask instructors for help in understanding relevant areas presented in the laboratory manual.
Introduction:
When a protein is treated with an alkaline solution of dilute copper sulfate (Biuret reagent), the Cu++ ions form a pink to violet coordination complex with the nitrogen atoms of the peptide bonds. The intensity of the colour produced is proportional to the number of peptide bonds and hence the quantity of protein present.
Materials:
• 1.0% (w/v) NaCl
• BSA (bovine serum albumin), supplied as 10 mg/mL in 1% (w/v) NaCl
• Biuret reagent
• a solution of protein and Biuret reagent for the preparation of an absorption spectrum
• unknown sample of BSA
Procedure:
• Set up a series of tubes for the preparation of a standard curve and for the unknown samples.
• Make the final volume of all tubes 1.0 mL using 1% (w/v) NaCl.
• Add 4.0 mL of the Biuret reagent to each tube and mix.
• Let the tubes stand for 30 minutes at room temperature.
• Generate an absorption spectrum of the solution supplied for this purpose.
• Determine the absorbance of prepared standards and unknown at the appropriate wavelength.
• Determine the concentration (in g/mL) of the unknown using information from your standard curve.
• Determine the amount of protein (in g) found in 8 mL of solution.
determine the volume in mL
95.0mL of a 2.05M KNO3 solution using a 4.00M KNO3 solution
To prepare your test tubes for the absorption spectrum, follow these steps:
1. Set up a series of test tubes for the preparation of a standard curve and for the unknown samples.
2. Make the final volume of all tubes 1.0 mL using 1% (w/v) NaCl. This means that you need to fill each test tube with 1 mL of the 1% (w/v) NaCl solution.
Next, you will need to add the Biuret reagent to each test tube:
1. Add 4.0 mL of the Biuret reagent to each test tube.
2. Mix the contents of each test tube thoroughly.
After preparing the test tubes, the next step is to let them stand for 30 minutes at room temperature. This allows the reaction between the protein and the Biuret reagent to occur.
In addition to the test tubes you prepared, there will be a solution supplied to you for the preparation of an absorption spectrum. You will need to generate an absorption spectrum of this solution using a spectrophotometer.
Now, for the measurement of the absorbance and determination of protein concentration:
1. Using a spectrophotometer, measure the absorbance of the prepared standards (different known concentrations) at the appropriate wavelength.
2. Also, measure the absorbance of the unknown sample at the same wavelength.
3. Use the absorbance values obtained to plot a standard curve, where the concentration is on the x-axis and the absorbance is on the y-axis. This curve will help you determine the protein concentration of the unknown sample.
4. Determine the protein concentration (in g/mL) of the unknown sample by reading the corresponding concentration value from the standard curve based on its absorbance.
5. To calculate the amount of protein (in grams) found in 8 mL of solution, multiply the protein concentration (in g/mL) of the unknown by 8 mL.
Remember to refer to any relevant information and calculations from your laboratory manual or any provided instructions or guidelines for accurate results.