C.B.S. Scientific HTLE-7002 - Manual (Page 13)

C. B. S. œ Scientific 13 HTLE-7002
D. Washing and Lyophilization
After digestion is complete the samples are then washed, lyophilized and dissolved
repeatedly to remove any ammonium bicarbonate and insoluble protein. The washed
peptides are solubilized in loading buffer and then applied to a thin layer cellulose plate. If
any salt or debris contaminate the sample it may cause peptides to streak or smear in the
first dimension (see below). This part of the procedure is crucial for preparing samples for 2-
D analysis, and must be done carefully if one wants to have clean, interpretable looking
maps.
To the digest (
∼70μl of ammonium bicarbonate solution) add 400μl of deionized water, freeze
and lyophilize (or just lyophilize if using a Speed-Vac). Add another 300
μl of water, vortex
well (at least 60sec) then relyophilize. Check to make sure there is no salt left, i.e. no fine
white powder visible on the sides of the tube. Count the lyophilized P
32 -labeled sample
before continuing with the final transfer step.
In order to be sure that all peptides loaded on the thin layer plate will be soluble in the buffer
chosen for the 1
st
dimension separation, a final transfer step is performed. Re-suspend the
sample in 300
μl of either pH 1.9 buffer, pH 4.72 buffer, or water (see below for explanation of
choices) and vortex well again. Centrifuge the sample for at least 2min in the microfuge at
RT, then transfer the supernatant, carefully avoiding any debris, to a new tube. If you are
preparing a C
14 or S 35 -labeled sample then remove 3
μl (1/100 th ), spot on a filter and count by
liquid scintillation. If any particulate material remains, resuspend the sample in 100
μl of
buffer, vortex and centrifuge again. Carefully remove the supernatant and check to see that
it is debris-free.
Lyophilize the sample until it is completely dry, then resuspend in 5-10
μl of the same buffer.
For P
32
-labeled samples estimate total yield by Cherenkov counting. The samples are now
ready for application on the thin layer plates and can be stored at 20
°C until use.
3.3 First Dimension: Thin-layer Electrophoresis
The first dimension of separation is the electrophoresis of the sample on a thin layer cellulose
plate. The optimal conditions are often determined empirically by using various buffers and
varying the power and time of the electrophoresis. We use three main volatile buffer systems
for separation in the first dimension for 20-30 min at 1.0kV:pH1.9 buffer, pH4.72 buffer and
pH8.9 buffer (named for their final pH). In general, more basic peptides are better resolved in
1.9pH buffer, more acidic peptides in 8.9pH buffer. At pH4.72 peptides with free carboxyl
groups such as glutamate are resolved well because the pH’s of the acidic chain are near
4.7. Included also is pH3.5 buffer, which is usually used for second dimension
electrophoresis separation of phosphoamino acids. We sometimes use this buffer in the first
dimensional of tryptic peptides and when plate purifying peptides phosphorylated in vitro. At
this pH
γ-P 32 ATP is well resolved from phospho-peptides.
There are no general rules that predict which system to choose. PH1.9 buffer is often used
as a starting point because most peptides are soluble in it and it resolve peptides very well.
The choice of buffer system will determine which buffer is used to resuspend the tryptic
digest during the final wash stage, except for pH8.9 when deionized water is used (see
below).