Question

BACKGROUND:

As reported in the journal, Blood (2016 vol 128:2097), the trifunctional, chimeric antibody, tri-31C2, is characterized by its ability to simultaneously bind to three different cell types: myeloma tumor cells, T cells, and accessory cells such as NK cells or macrophages.  

As a research associate in the lab, you have been asked to purify a large quantity of tri-31C2 from the supernatant (media) of its hybridoma in tissue culture. Although you are purifying this antibody from tissue culture media and not from serum, the purification procedure still involves SAS fractionation followed by anion-exchange chromatography.

Address the following two sets of matching questions regarding these methods.

Match each of the following steps in SAS precipitation with its purpose:

1. add 0.5 ml SAS to 0.5ml culture medium

a.to improve precipitation of the protein

b.to remove salt from sample, to bring sample to pH 8.0

c.to be sure IgG is positively charged

d. to collect precipitated IgG (crude) as a pallet

e.to precipitate IgG (crude) out of solution (at 50% SAS)

2. centrifuge the precipitated sample

a.to improve precipitation of the protein

b.to remove salt from sample, to bring sample to pH 8.0

c.to be sure IgG is positively charged

d. to collect precipitated IgG (crude) as a pallet

e.to precipitate IgG (crude) out of solution (at 50% SAS)

3.dissolve precipitate in water, dialyze against tris buffer, pH 8.0

a.to improve precipitation of the protein

b.to remove salt from sample, to bring sample to pH 8.0

c.to be sure IgG is positively charged

d. to collect precipitated IgG (crude) as a pallet

e.to precipitate IgG (crude) out of solution (at 50% SAS)

Answer 1

1. add 0.5 ml SAS to 0.5ml culture medium

e. to precipitate IgG (crude) out of solution (at 50% SAS)

2. centrifuge the precipitated sample

d. to collect precipitated IgG (crude) as a pallet

3.dissolve precipitate in water, dialyze against tris buffer, pH 8.0

b. to remove salt from sample, to bring sample to pH 8.0