Dye modification of lipids
Malmstadt Lab Protocol
Title: Modification of Phosphotidylethanolamine lipids with NHS-ester dyes
Author(s): Li, Malmstadt
Origination Date: 1/9/08
Last Modification Date: 1/9/08
Reagents Needed
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Methanol
Chloroform
Phospotidylethanolamine lipid (e.g. DMPE from Avanti)
NHS-ester dye (e.g. AlexaFluor 488 succinimidyl ester from Invitrogen)
One large (20 cm  20 cm) 250-µm-thick silica gel thin layer chromatography plate
Ether
Equipment Needed
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Water bath
Stir plate
Fume hood
Thin layer chromatography tank
UV illuminator
Vortexer
Centrifuge with capacity for 50 mL glass tubes
2 50 mL glass centrifuge tubes with caps
Rotary evaporator (optional)
Procedure
1. Reaction of lipid and NHS-ester
a. Dissolve 2 mg of lipid in a 3:1 (volume:volume) mixture of methanol and chloroform in a glass vial.
It will be necessary to heat the solvent in a 60 ºC water bath in order to fully dissolve the lipid.
b. Add 1 mg NHS-ester dye to lipid solution. It may be necessary to transfer the lipid solution to the
tube in which the dye was shipped, since it is very difficult to measure and transfer this small
amount of powdered solid. Once the dye is fully dissolved, return the solution to the glass vial,
add a micro stir bar, and stir rapidly for one hour at room temperature.
c.
Incubate reaction solution at 4 ºC overnight.
2. Separation of modified lipid and unreacted dye
a. Wash a large chromatography plate with developing solution (in fume hood)
i. Pour ~80 mL of developing solution (65:25:4 chloroform:methanol:water by volume) into
the bottom of the thin layer chromatography tank.
ii. Place TLC plate into tank. Cover tank and allow the developing solution to be drawn
entirely up the plate.
iii. Remove plate from tank and pour off any remaining developing solution into waste bottle.
Allow TLC plate to dry in fume hood for ~1 hour.
b. TLC separation of lipid and dye (in fume hood)
i. Streak a thin line of reaction mixture (from step 1c.) onto TLC plate, approximately 2 cm
from the bottom. Allow to dry for ~20 min.
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Dye modification of lipids
ii. Pour ~80 mL of developing solution (as in step 2ai.) into the bottom of the TLC tank.
iii. Place TLC plate into tank. Allow the developing solution to be draw up the plate until
there is a clear separation between lipid and dye lines, as illuminated by UV. The lipid
line should migrate up the plate more quickly.
iv. Remove the plate from the tank and allow to dry in the fume hood for ~1 hour.
3. Extraction of reacted lipid from TLC matrix (in fume hood)
a. Scrape the silica gel along the lipid line from the TLC plate into a glass vial.
b. Add ~10-15 mL of eluting solvent (1:1:1 chloroform:methanol:ether) to the scraped silica matrix.
Vortex vigourously, and centrifuge to pellet silica. Reserve supernatant
c.
Repeat step 3b twice and pool supernatants.
d. Dry pooled supernatants in a rotary evaporator or under a nitrogen stream.
See pg. 251 of Kates’ Techniques of Lipidology for a more detailed elution protocol.
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