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Preparation, Analysis and Use of an Affinity Adsorbent 131
3.8. GST Activity Assay
GST activity assays are performed on the lysates, flow-through and elution
fractions in order to determine the concentration of the GST–ZnF molecule and
perform a mass balance.
1. Add 10 μl of sample to 1 ml of GST enzyme activity assay reagent and mix by
inverting the sample four times.
2. Perform a rate analysis at OD 340 nm to detect the GST-mediated reaction of CDNB
with glutathione. Dilute GST–ZnF samples to concentrations less than 2 mg/ml so
that the change in activity over time is linear.
3.9. SDS–PAGE
Lysate and protein samples were analyzed by SDS–PAGE using NuPAGE
Novex Bis-Tris 4–12% Gels run in an XCell Mini-Cell (Invitrogen, UK).
1. Incubate 10 μl of protein containing samples with 10 μl of protein gel loading
buffer for 5 min at 95°C.
2. Load the protein sample aliquots into the gel wells. Up to 10 samples can be run
simultaneously.
3. Run gels in MOPS SDS running buffer (Invitrogen) for 50 min at 200 V.
4. Remove gels from the cartridge and stain for 2 h using SDS–PAGE staining
solution.
5. De-stain overnight in SDS–PAGE de-staining solution. Gels were photographed
using the EDAS 290 utilizing a visible light illuminator. When densitometry studies
were performed, the images created by the EDAS 290 were analyzed using Kodak
Digital Science 1D Image Analysis Software and compared to protein markers of
known concentration.
4. Notes
1. Glutathione should be stored at 2–8°C.
2. CDNB IS TOXIC (by inhalation, contact with skin or if swallowed) and should
be handled in a fume hood.
3. This first washing stage is to remove ethanol that helps to preserve the adsorbent.
Extensive washing usually requires at least three wash/bed settle stages in batch
mode or at least 10 column volumes if washed in a chromatography column. In
batch mode, the smell and a change in resin morphology indicate that the ethanol
has been removed. For a chromatography column, the OD 260 nm of the stream
exiting the column will be constant. If ethanol is present, the binding capacity of
the adsorbent for the target may be affected.
4. Sodium borohydride acts as a reducing agent and assists in stabilizing the bonds
between the spacer arm and solid-phase adsorbent.