C.B.S. Scientific HTLE-7002 - Manual (Page 27)

C. B. S. œ Scientific 27 HTLE-7002
B. Preparing the Chromatography Tank & Chromatography
Prepare the chromatography buffer(s) and pour enough into a tank so that it is about 2-2.5cm
deep. Make sure that the tank is level, then measure the depth. You need enough liquid to wet
the bottom of the cellulose side of the plate while it is on a slant, roughly 1cm from the bottom. It
is best to do this the day before chromatography so the buffer completely equilibrates with the
gas phase of the system. To accelerate the equilibration step some people have placed
Whatman 3MM paper along the inside of the tank and allow the buffer to climb up the walls.
Regardless, the tank should be kept undisturbed with the lid on for several hours prior to each
run. The buffer will last for several months in the tanks or storage bottles if tightly capped. It is a
good idea to keep a log of how many plates are run in each buffer tank and how long since the
last buffer change. Since each buffer contains pyridine it is best to use amber glass bottles for
storage.
Before chromatography spot a drop of the same dye marker (0.5
μl) to a spot adjacent to the
sample origin but over in the left-hand margin (see diagram of plate stencil). This will act as a
colored marker for the second dimension and a reference for calculating the relative mobilities of
peptides. Note that the dye may partially resolve into individual yellow and blue components. To
begin chromatography, carefully remove the lid of the tank and place the marked plates bottom
down into the tank. They should be placed at roughly an 80
° angle with the top of the plate
resting on the walls of the tank. You should be able to load up to eight plates per tank if you use
glass bottles to form a center aisle. Once the plates are set quickly replace the lid and make sure
an air-tight seal is formed. Chromatography is usually carried out for 6-8hr until the buffer front
has reached about 3cm from the top of the plate. The period needed to chromatograph plates
will vary depending on the buffer used and the separation desired. Do not disturb a run or enter
the tank while a run is in progress. When complete, remove the lid and carefully lift each plate
from the tank, making sure not to drip any buffer on plates remaining inside. Air dry the plates in
a fume hood until you can no longer smell any residual buffer (this usually takes about 60min). If
peptides are to be extracted from the plates for further analysis then we do not recommend
drying the plates by baking in an oven, but otherwise they can be. Once dry the peptide maps
are completed and ready for fluorography (C
14
or S
35
) or autoradiography (P
32
or I
125
).
3.5 Phosphoamino Acid Analysis
The method for 2-dimensional phosphoamino acid analysis (PAA) has been previously described
in detail (3). Their protocol has been updated below with particular emphasis on using the HTLE-
7000 system. Begin with material that was prepared in section IIb (TCA-precipitated proteins
before or after oxidation in performic acid). Resuspend the sample using 50-100
μl of constant
boiling HCl (5.7M), vortex, then centrifuge briefly and incubate at 110
°C for 60min. If using
Eppendorf microfuge tubes we find it necessary to tape two test tube racks firmly on top of each
other so the lids of the tubes remain closed. One can also use Sarstedt screw-top tube which
remain closed without tapping. It is essential to note that this is a partial amino acid hydrolysis
and that incubation times significantly longer than 60min will result in excess liberation of free
phosphate from phosphoamino acid residues.
After hydrolysis, dry the sample by lyophilization in a Speed-Vac (equipped with an NaOH trap to
collect acid) and determine Cherenkov counts remaining. Resuspend the hydrosylate in 5-10
μl
of pH 1.9 buffer (same buffer used for peptide mapping) which contains 15 parts of buffer to 1
part of cold PAA standards (1.0mg/ml each phosphosyrene, phosphothreonine and
phosphotyrosine in deionized water). You can analyze 4 samples on one plate using the PAA
stencil as a guide (see appendix). Select and mark a TLC plate as mentioned before in section
IIa on peptide mapping.