Extraction Procedure

Ideally this should be done in red or gold light, but at the very least it should be done in subdued lighting (turn the lab lights off) and in lightproof (brown) microcentrifuge vials. All chemicals should be of the highest quality.

Fig. 2. Examples of HPLC chromatograms using the methods described in the text. Solid lines show the UV absorbance at 351 nm, and dotted lines the cpm measured with an on-line radioactivity detector. (A) A mixture of six retinoid standards separated out into individual peaks with different elusion times. Peak 1 = retinal, peak 2 = tRA, peak 3 = retinol, peak 4 = 13-cis-RA, peak 5 = didehydroretinol, peak 6 = 4-oxo-RA. The dotted line marks the cpm of [3H] tRA, which was also added to the mixture and this coeluted with the cold tRA. (B) The retinoid extracted from a whole 10.5-d mouse embryo. The same six peaks of known standards are marked. Only retinol

Fig. 2. Examples of HPLC chromatograms using the methods described in the text. Solid lines show the UV absorbance at 351 nm, and dotted lines the cpm measured with an on-line radioactivity detector. (A) A mixture of six retinoid standards separated out into individual peaks with different elusion times. Peak 1 = retinal, peak 2 = tRA, peak 3 = retinol, peak 4 = 13-cis-RA, peak 5 = didehydroretinol, peak 6 = 4-oxo-RA. The dotted line marks the cpm of [3H] tRA, which was also added to the mixture and this coeluted with the cold tRA. (B) The retinoid extracted from a whole 10.5-d mouse embryo. The same six peaks of known standards are marked. Only retinol

1. Collect batches of embryonic tissue on ice. Not <500 mg can usually be used. They can be stored for short periods at -70°C to allow the collection of enough material.

2. Add an equal volume of ice-cold stabilizing buffer and sonicate. Stabilizing buffer = 5 mg/mL ascorbic acid + 5 mg/mL Na3 EDTA dissolved in PBS, with pH adjusted to 7.3 with NaOH.

3. Take 10 |L of homogenate for protein estimation, so that at the end the amount of retinoid can be given in ng/mg protein. Alternatively, use this sample for DNA determination, and express the data in ng/|g of DNA.

4. Add a known amount, 1-2 nC, of [3H] tRA, so that the recovery ratio can be determined and the tRA peak identified on the chromatograph.

5. Extract in 2 vol of extraction solvent. Extraction solvent = ethyl acetate:methyl acetate 8:1 + 50 |g/mL butylated hydroxytoluene as an antioxidant. Extract for 20 min while continuously mixing on a vibromax.

6. Separate the solvent phase by microcentrifugation at low speed. Keep the solvent phase.

7. Repeat the extraction on the homogenate, and separate again by centrifugation. Remove the solvent phase, and pool it with that from the first extraction.

8. Dry down the combined solvent phases under a stream of nitrogen.

9. Resuspend the extract in 100 |L methanol, and microcentrifuge at high speed to remove the particulate matter. This is now ready to be injected onto the HPLC column.

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