Question

Can you please desribe what steps need to be taken to:

Protein 33 from the complex mixture has been found to be critical to the academic interests of for reasons that have not been disclosed by the administration. Your assignment is to purify protein 33 in the most efficient way possible. You should endeavor to:

i) purify the protein to homogeneity with respect to SDS-PAGE analysis (i.e. no other visible bands).

ii) lose no more that 20% of the original activity.

iii) achieve purification in the least number of person-hours per unit of activity.

Using The ProtLab program, which may be accessed from web site - http://www.leeds.ac.uk/bionet/compend/bnt11pst.htm.

( http://home.btconnect.com/agbooth/archive/ )

Answer 1

Affinity chromatography is a biophysical method used to separate molecules based on a specific binding interaction between an immobilized ligand and its binding partner.

• First of all, take the given complex sample in a buffer solution.

• Take the protein specific antibody, and bind it to a gel matrix using secondary antibodies against that antibody or any chemical, which can bind it.

• Equilibrate the affinity medium with binding buffer

• Apply sample, which contains membrane receptors

• Desired molecules bind specifically to the ligand (protein 33 polypeptide bound to the gel matrix)

• Unnecessary proteins and remnants elutes first

• Then, elute the target protein by changing the conditions to favour the elution process. For example, by changing the pH, ionic strength, or polarity

• And collect the target protein in a purified form