Issel Lim
Protocol for Protein Biochemistry Day 3
Experiment IIA/IIIB: “APTG Affinity and PD-10 Gel Filtration Chromatrography”
APTG Affinity Chromatography
Preparation
After thawing the DEAE/AF Load fraction, it was put on ice. The affinity (1.5mL) column on
the ring stand was filled with 1mL of 50% APTG-agarose slurry. The affinity column was
equilibrated with 2.5mL of column buffer, in 0.5mL increments. 1.5mL tubes were labeled with
“FT#1”and “FT#2” for the flow-through solution, “Wash#1” and “Wash #2” for the washes, and
“AF#1,” “AF#2,” and “AF#3” for the affinity fractions. After microfuging the protein sample
for 20 seconds, the supernatant was put into the column and collected in a 1mL fraction in the
tube labeled “FT#1.” The column was washed with 0.5mL increments of a total 2.5mL of
column buffer. 0.5mL of solution after wash went to the “FT#2” tube. The column was plugged
just before the liquid reaches the bed top.
Elution
The bound protein was eluted from the column with 3mL of elution buffer in 1mL increments in
the tubes “AF#1,” “AF#2”, and “AF#3.”
Analysis
The enzyme activity in the FT tubes and the AF fractions was checked using a qualitative ßgalactosidase assay. The highest activity/affinity fraction was labeled “AF before PD-10.” 25
microliters of it was pipetted into a 0.5mL Eppendorf tube on ice. 25 microliters of the first two
FT fractions went into 0.5mL Eppendorf tubes put on ice.
PD-10 Gel Filtration Chromatography
Preparation
During the APTG Affinity elution, the PD-10 column was equilibrated by cutting off the tip,
plugging it with a plastic tip, and discarding the original buffer. Then 5mL of column buffer
rinsed the column five times (25mL total). The fractions eluted from the PD-10 column were
collected in six 1.5mL Eppendorf tubes “#1 -- #6.”
Elution
The remaining “AF before PD-10” was loaded into the PD-10 column, then 0.1mL of column
buffer ensured that all of the sample went through the tube. The column was eluted six times
with 1mL of column buffer in tubes #2 -- #6.
Analysis
The PD-10 fractions were qualitatively assayed for ß-gal activity. The highest activity fractions
were put into “AF after PD-10” tubes. 50 microliters of this was saved in a 0.5mL Eppendorf
tube, and all samples were placed on ice. A quantitative assay was then performed on the “AF
after PD-10” fraction. The total activity of the “AF after PD-10” fraction was calculated, along
with the yield of ß-gal after each purification step.