Question

You have isolated a lyophilized (in powder form) protein from a particular cell line. You have at least a gram of the material and a completely equipped laboratory including instruments, supplies, and reagents. List the steps you would take to determine the absorptivity of your protein in units of mL/mg·cm.

Answer 1

Materials provided: a lyophilized protein 1 gm

Aim : to determine the absorptivity of the protein

Steps involved:

1. Get the lyophilized protein sample and keep it at room temperature for some time.

2. Then centrifuge the lyophilized protein sample(kept at Room temperature) so that the protein get accumulated at the base of the cetrifuge tube.

3. After that resuspend the protein sample in a suitable buffer (say phospate buffer) by adding a suitable volume (e.g 1mg/ml)

4. After that Follow a specific protein estimation protocol like Lowry's method or Bradford's method. use suitable aliquots of known concentration and read their absorbance at the given wavelength (e.g. 280nm).

5. then use the following formulae

A = e C L

where A = is the absorbance, e is the absorptivity or Molar extinction coefficient, C is the concentration, L is the length of path

from the above formulae,

we get e= A/CL

A is known , C is also known to you , L is fixed for a cuvette

Hence we can easily calculate the value of e in ml/mg. cm