220.127.116.11. Isolation and identification of Dufour's biosynthesis products.
The glands and incubation media are extracted in 350µl dichloromethane for 24 h after incubation and then subjected to TLC. To isolate Dufour's gland biosynthesis products, carry out TLC of the extract using silica gel coated plates (polygram Sil G). The radioactivity of the various TLC fractions can be determined by a phosphor imager (IP Autoradiography System). Quantification of the radioactive fractions (Rfs) is achieved by comparison of its radiation to a standard radiation curve generated using different doses of radioactive acetate.
TLC separation is performed in two successive steps:
- Run the TLC plate using hexane to separate the components.
- Air-dry the plate in a fume hood.
- Subject to a second separation in a mixture of hexane: diethyl ether: acetic acid (70:30:1) (v/v), as the running solvent.
- Identify the various lipid classes by comparing their Rf values with those of co-chromatographed standards (unsaturated).
- Visualize by iodine vapour (in pre-prepared saturated
In case of Dufours gland, suitable standards are: cis-9-tricosane
(hydrocarbons); oleic acid (fatty acids); 11-eicosenol (alcohol); palmitic acid
myristhyl ester and palmitoleic acid stearyl ester (esters); trinervonin and triolen
(triglycerides). This procedure can be validated by using unlabelled products
followed by GC/MS analysis as described below.
- Incubate Dufour’s glands as described in section 18.104.22.168. using cold acetate as a precursor (0.3 mg/ml).
- Apply the glandular extracts on TLC along with commercial TLC standards (as above).
- Perform TLC separation as previously described and allow the plate to dry.
- Cut off the part of plates containing the commercial standards and expose it to iodine vapour to prevent modifications to compounds in the sample lanes designated for GC-MS analysis.
- Mark the position of the standards.
- In the sample lanes scrape the silica gel of the areas corresponding to standards on the TLC, into a glass vial and extract with chloroform, evaporate to dryness, reconstitute in dichloromethane.
- Concentrate using N2 and subject the product to GC-MS to identify relevant peaks as described in section 2.2.4.
Pros: The obvious advantage of the above described system is in its ability
to study the activity of the exocrine gland detached from its original
controlled environment. By manipulating the environment of the gland, factors
and mechanisms regulating glandular activity can be isolated. This technique
enables the researcher to separate between the effects of social regulation and
possible physiological constraints of any studied gland.
Cons: However, the isolated gland function may not necessarily represent the full range of glandular functions as some of these may be dependent on unknown precursors received from elsewhere in the body of the bee. Thus, it is advisable to compare in vitro function of the gland with in vivo labelling studies, followed by glandular extraction and determination of de novo synthesized products.