Activity 1: Spectrophotometry: using a calibration curve




Activity summary

What you must remember :
Skills linked to the curriculum:

Compétences Capacités à maîtriser
COM
APP

Savoir expliquer un protocole.
Utiliser du vocabulaire spécifique.
APP
ANA
Savoir exploiter une courbe d’étalonnage.


Download activity

word  pdf





Documents

Document 1: BSA protein

Bovine serum albumin (also known as BSA or "Fraction V") is a serum albumin protein derived from cows. It is often used as a protein concentration standard in lab experiments.



Source: https://en.wikipedia.org


Document 2: Spectrophotometry measurements with proteins using Brilliant Blue dye (Bradford method)

The dye (Bradford reagent) associated with BSA portein has an absorption spectrum maximum at 595 nm. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the concentration of protein present in the sample.

The procedure for Bradford protein assay is very easy and simple to follow. It is done in one step where the Bradford reagent is added to a test tube along with the sample. After mixing well, the mixture almost immediately changes to a blue color and the absorbance can be read at 595 nm using a spectrophotometer.

Source: https://en.wikipedia.org


Document 3: Procedure for standard assay

Procedure (Standard Assay, 20-150 µg protein; 200-1500 µg/mL)

- Prepare a series of protein standards diluted with 0.15 M NaCl to final concentrations of 0 (blank = NaCl only), 250, 500, 750 and 1500 µg/mL. Also prepare serial dilutions of the unknown sample to be measured.
- Add 100 µL of each of the above to a separate test tube
- Add 5.0 mL of dye to each tube and mix by vortex, or inversion.
- Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube which contains no protein.
- Wait 5 minutes and read each of the standards and each of the samples at 595 nm wavelength.
- Plot the absorbance of the standards vs. concentration.

Source: https://en.wikipedia.org


Document 4: BSA protein calibration curve


Figure : Calibration curve - generated from BSA samples measured at 595nm

Source: GE Healthcare Life Sciences Spectrophotometry Handbook



Acquiring vocabulary

English French
lab experiments

wavelength

une mesure 
  un essai 
  un échantillon 
   un colorant
 (protein) bound dye  
 the process  
  absorbance 
  une courbe d'étalonnage 
 reagent  


Presentation
Prepare a short 2min presentation:
Using the knowledge acquired in chemistry class and document 3, recap the procedure carried out during spectrophotometry measurements?

Questions
1. At which wavelength were measurements carried out on BVA protein? Explain.
2. According to document 2, how was this value chosen?
3. An unknown sample has an absorbance of A = 0,3, work out the BSA concentration in the sample.


Last modified: Sunday, 3 February 2019, 11:16 AM