Bio-Rad Human Metabolic and Hormone Assays - Manual (Page 19)

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5. After the incubation , centrifuge the extraction tubes in a
microcentrifuge at 8,400 x g for 10 min (±1 min).
6. Add 50 µl neutralization buffer (1 M Tris, pH 7.5) to each of the tubes
labeled for neutralization.
7. Before transferring the extract into neutralization buffer, prewet each
pipet tip by pipetting up and down 2–3 times in a small amount
(~1 ml) of extraction buffer. This should be done in a clean 1.5 ml
tube.
Note: Do not prewet the pipet tip in the extracted sample, as this
may resuspend a portion of the compacted pellet.
8. Without disturbing the pellet, transfer 50 µl extracted supernatant
to the corresponding neutralization tube (containing 50 µl of
neutralization buffer), cap and, vortex.
9. Add 50 µl sample dilution buffer 2 to the neutralized sample. Final
sample dilution is 1:30.
10. Store samples on ice for immediate testing. Alternatively, store them
at –70°C for up to one month.
Materials Vendor/Part # Amount required (ml)
1 M Tris-HCI, pH 7.5 Fisher/NC9053751 As needed
Table 6. Preparation of IGF neutralization buffer.