logK - another procedure

Prepare ahead of time

Add 1xPBS and 1,9-decadiene to a vial and vortex to saturate the decadiene with water and vice versa. Let stand. Refrigerate? It takes about an hour for the layers to separate.

Dilute your samples ahead of time as well. Volumes cannot be accurately dispensed below 5μL, so dilute concentrated stocks in a stepwise manner. Stock concentrations of 200μM work well for logK, PAMPA, and fSol.

The assay

Your compound of interest will be partitioned between decadiene (a hydrocarbon) and 1XPBS. The logarithm of the ratio [sample]decadiene:[sample]1XPBS is logK.

logK experiments don’t need to be a full mL, but should be at least 500micL. Matt uses 600micL.
300micL each of 1xpbs and decadiene

First, place 3micL of your 200micM sample in an empty 1.5mL eppendorf.
Vortex your samples right before dispensing.
Then, place 300micL each of 1xpbs and decadiene into one of the eppendorfs from your pre-saturated layers.
Now, we need to thoroughly mix.
First, vortex. Matt’s vortex has a timer. Do 20 minutes.
Next, sonicate, for 20 minutes.

The eppendorf is now centrifuged for 20 minutes on maximum speed (14,000 rpm in our case).

Our layers are now separated.

Next, you will need to be very careful when separating the layers. Slight contamination of the aqueous with decadiene is going to be a big problem.

So, transfer 100 or 150micL of each layer into a separate eppendorf. It doesn’t matter, but you should collect the same volume of each. The top layer is pretty easy. Pipet from the middle of the layer, and do not let the tip touch the sides of the eppendorf.

To pipet the water layer, pull a tiny bit of air into the tip, and expel once you are in the center of the aqueous layer. This will prevent decadiene layer from being in the aqueous. Once you have pulled your volume, consider wiping the outside of the tip with a kimwipe before releasing into the separate eppendorf.

Now, we need to evaporate the solvent overnight. You can use the genevac, or you can do a blowdown.

The next day, the decadiene layer is ready to go. Add 100micL of DMSO, and this can go directly on the orbitrap.

The PBS layer should be taken up in 100micL DMSO, then vortex, then sonicate, then centrifuge. Withdraw some DMSO without disturbing the pellet and this can go on the orbitrap.

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