Reference no: EM132833110

BC2013 Principles of Biochemistry - James Cook University

Practical - Standard Curves

Learning Outcome 1: Practical skills - handle pipettes competently and understand the use of UV-VIS spectrophotometers

Learning Outcome 2: Recognise the relationship between absorbance, concentration and molar absorption coefficient (extinction coefficient) as stated in the Beer-Lambert law

Learning Outcome 3: Recognise that dyes can have spectral properties that allow us to quantitate molecules in solution

Learning Outcome 4: Prepare and interpret a standard curve

Learning Outcome 5: Rearrange the Beer-Lambert law to calculate an unknown component

Preparation

- Revise practical 1 from BM1000 - Spectrophotometers and Pipettes and the first practical (pipetting).
- Revise CH1001 material on Beer's law and molarity.
- Familiarise yourself with units of volume, mass and concentration and the equations that interconvert them.

Make sure that you have shown a demonstrator a completed Gantt chart of your workplan for the practical before your sign in.
a) Pipettes
• Have a good look at a pipette, practice pushing the button in and releasing
• Always use a tip - don't allow liquid to get up the barrel
• First stop only when drawing liquid up
• Second stop to get all the liquid out
• Never overwind a pipette - look at the numbers on the side or the button, don't try to exceed the range of the pipette
• Don't drop the pipettes - they are (EXPENSIVE $400) precision instruments

b) Spectrophotometers
• Have a look inside the sample chamber of a spectrophotometer.
• Which direction does the light come from?
• Which side is the detector?
• Notice the width of the cuvette = 1 cm
• How is wavelength changed?
• How is the instrument calibrated?

Part A Create a standard Curve
1. Label 5 sets of 3 microfuge tubes:
25a, 25b, 25c; 20a, 20b, 20c and so on for 25, 20, 15, 10, 5 μg standards.
2. Label two tubes as blanks: B1, B2
3. Prepare your standards each in a volume of 50 μl. Aliquot the amounts as per Table 1 starting with H2O. Notice that the amount of protein in the tubes is changed but the volume is kept constant. The blanks have dye solution but no protein.
4. To each of the microfuge tubes add 1500 μl of the dye solution.
5. Mix all tubes well by carefully inverting them several times and allow to stand at room temperature for at least 5 minutes (they must be read within an hour).
6. Transfer B1 into the one of the cuvettes.
7. Ensure the spectrophotometer is set to 595 nm.
8. Place the cuvette inside the sample chamber, check that the clear sides of the cuvette are facing the same direction as the light.
9. Hit ""zero" to set this as the zero point (see instructions on the machine).
10. Pour the solution in the cuvette back into the microfuge tube and keep it in case you need to read it again. Repeat for B2. The values should be within 0.02 absorbance units of each other. If not you will have to prepare them again.
11. Using a 1 ml pipette, transfer the 5 μg standard into the second, sample cuvette and read the absorbance. Record the value. Pour the solution back into the microfuge tube and keep it in case you need to read it again.
12. Repeat for all the samples in order of increasing amount of protein (i.e. from lighter to darker solutions)
13. Plot the reading on a graph with amount of protein (not volume or concentration!) on the x- axis and absorbance on the y-axis. Ensure you label all axes, the graph should have a descriptive title (-1/2 mark for "Absorbance vs. Amount of Protein" or "Standard Curve").
14. Draw a line of best fit through the origin - check with a tutor.

Part B Find the concentration of your unknown sample
The sample you have been given is a sample of the same protein as used for the standards (BSA).

1. You will need to dilute it so that it falls within the range of the standard curve, but the sample volume must be 50 μl. You will need to read at least three replicates of the final sample dilution.
(Hint: start by diluting by half and see if the reading is within range, adjust dilution factor accordingly).

2.
a) Calculate the amount of protein in the original sample in milligrams per milliliter.
i.e. Standard curve tells us how many μg per 50 μl Multiply by dilution factor and convert to mg/ml
b) The molar mass of BSA is 67000 gmol-1 (molecular mass of 67 kDa or 67 000 Da). Calculate the molarity of your original sample.

3. Calculate the extinction coefficient for BSA bound to Coomassie blue at 595 nm using the data from the 10 µg standard.

6. Report
The report for this workshop is marked out of 8 and should contain the following information (rubric on next page):

Part A
• A table of results.
• The standard curve you obtained from your results, correct title, correctly labeled axes.

Part B
• The amount of protein in your unknown sample including a full calculation showing the dilution factor you used. Remember to include units!
• The extinction coefficient of BSA:Coomassie complex including a full calculation. Remember to include units!

Questions
a) Why are readings taken in triplicate?
b) How should outlier points be treated in a standard curve?

Attachment:- Standard Curves.rar