Thin layer chromatography (TLC)

Equipment:
50 mL beaker
ceramic hot plate
chemical fume hood
20 mL glass bottle or other similar vessel with closable lid
forceps

Consumables:
18 gauge needle 
10 mL syringe
glass pipettes and pipette bulbs
aluminum foil
10% w/vol phosphomolybdic acid in ethanol
TLC plates
2:1 diethyl ether:hexanes

Preparation of 10% w/vol phosphomolybdic acid in ethanol:
weigh out 10 g of solid phosphomolybdic acid and add it to a clean 100 mL glass bottle with a wide mouth (ex: Fisher Scientific: FB800100). Top off the the bottle to 100 mLs with absolute 200 proof ethanol. Close the top of the bottle and mix by inversion until the phosphomolybdic acid is fully resuspended. Store protected from light at 4 C.

Preparation of the TLC plate: using scissors, cut out a roughly 3 inch by 2 inch piece of an aluminum-backed TLC plate, taking care to not cause the coating to flake off. 

Preparation of your 2:1 mix of diethyl ether:hexanes: No more than an hour prior to running your TLC, make up the 2:1 mix of diethyl ether and hexanes. for 15 mL of running solvent, combine 10 mL of diethyl ether and 5 mL of hexanes. Store in a glass bottle with a closable lid.
Notes: a) ether usually comes in a bottle with a stopper top. To pull it out of the bottle, use a 10 mL syringe with a 18-gauge needle  to pierce the stopper and take up the needed amount of ether. b) hexanes will melt plastic. only use glass pipettes to take up hexanes and only store hexanes in glass vessels. c) perform all hexanes and diethyl ether work in a properly functioning fume hood.

Procedure, as performed for manuscript:
The components of the enzymatic reaction are mixed at the following concentrations in an Eppendorf tube:
4 µM final enzyme
1.6 mM 4-HNE
1.6 mM NADPH
20% w/v PEG-800
in 200 µL final volume of phosphate buffered saline (PBS)

The reaction is allowed to proceed for an 1 hour at room temperature (22 C) with no agitation.

While the reaction is running, label your pre-cut TLC plate by drawing a line in pencil across the narrow part of the plate roughly 1/4th of an inch above the bottom. draw evenly spaced spots across this line and label them, also in pencil. 

Once the reactions are complete, spot 6 µL of each reaction onto the appropriate pre-labeled spot and let dry completely. 

Take the 50 mL beaker and set it up in the fume hood. fill about a 1/8th of an inch of the bottom of the beaker with the 2:1 diethyl ether:hexanes mixture. cover the beaker with a piece of foil. 

Using forceps to avoid unintended chemical transfer to the TLC plate, gently grab the plate and place it into the 50 mL beaker in such a way that the TLC plate is vertically leaning against one side of the beaker with the spotted portion of the plate 1/8th of an inch above the running solvent (ether and hexanes mixture). take care that the spots DO NOT TOUCH the ether:hexanes. 

Cover the reaction chamber with foil and watch the solvent line rise through the TLC plate. do not let the solvent line run off the plate. 

Once the line reaches about 1/4th of an inch from the top of the plate, take off the foil and using forceps pull out the TLC plate. Let dry in the fume hood (1-5 minutes).

Open the 10% w/vol phosphomolybdic acid bottle. Use forceps to  dip the dry TLC plate into the solution in one gentle and smooth motion. This should  take 10 seconds. Pull the plate out of the solution and lay it in the fume hood to dry for a few minutes.

Turn on the ceramic hot plate to the highest heat and place a piece of foil on it to protect the ceramic from phosphomolybdic acid stains. 

using forceps, place the TLC plate aluminum side down onto the hot aluminum foil on the hot plate. watch the TLC plate as it changes colors. it should go from bright yellow to light yellow and then dark/black spots will begin to appear. This process will take between 15 seconds to a minute. Do not overheat as that will overexpose the spots.

Once done, pull the TLC plate off the hot plate and let cool.