Bio-Rad Human Metabolic and Hormone Assays - Manual (Page 18)

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Cell culture media
1. Collect cell culture supernatants and centrifuge at 1,000 x g for 15 min
at 4°C. For cell lines cultured in serum-free media, collect samples and
add BSA as a carrier protein to a final concentration of at least 0.5%.
This is done to stablize protein analytes and to prevent adsorption to
labware.
2. Transfer to a clean polypropylene tube. If cellular debris or precipitates
are present, centrifuge at 10,000 x g for 10 min at 4°C.
3. If high levels of analyte are expected, samples can be diluted in culture
media. Optimal dilution factor must be determined by the end user.
Supplement serum-free media with at least 0.5% BSA final.
4. Assay samples immediately or aliquot and store at –70°C.
Note: Compatibility of a given media type with the assays must be
validated by the end user.
Sample Extraction for IGF-1 and IGF-2
Prepare fresh extraction and neutralization buffers immediately before use.
1. For each sample label two 1.5 ml polypropylene microcentrifuge tubes,
one for extraction and one for neutralization.
2. Pipet 20 µl sample into the corresponding 1.5 ml extraction tube.
3. Add 180 µl extraction buffer (12.5% 2 N HCl and 87.5% ethanol) to
each extraction tube for a 1:10 dilution.
4. Cap and vortex each tube. Incubate for 30 min (±2 min) at RT. After
the initial 15 min incubation, vortex the tubes and allow to sit for the
remaining 15 min.
Materials Vendor/Part # Amount required (ml)
5N HCI Fisher/NC9053751 0.500
DI water, 18 mΩ Super - Q purified N/A 0.7500
Ethyl alcohol, denatured Fisher/50-980-458 8.750
Table 5. Preparation of IGF extraction buffer.