C.B.S. Scientific HTLE-7002 - Manual (Page 10)

C. B. S. œ Scientific 10 HTLE-7002
3.2 Preparation of Samples
Note: Use radioactive solutions in accordance with the safety regulations of your
institution, contact your safety officer. This method is useful for mapping either C
14 , S 35 or
P 32 -labeled proteins. For phosphorylated samples a small portion can be used for phosphoamino
acid analysis while the remainder can be used for phosphopeptide mapping. The only difference
between C
14
or S
35
labeled samples and P
32
-phosphorylated samples is that you can monitor the
recovery of the P
32
-label at each step by Cherenkov counting. For C
14
or S
35
-labeled samples
you must proceed blindly until the final transfer step. The entire protocol will usually take 3-4
days, but can be accelerated if necessary. For many steps the timing is flexible, and there are
convenient points to stop overnight.
A. Elution from Polyacrylamide Gels
The most common way to prepare samples is to localize either protein “bands” from one-
dimensional (Laemmli) gels, or “spots” from two-dimensional gels. This can be accomplished
by either staining the gels in Coomassie blue stain or by autoradiography. Treating gels with
fluors to help detect weak signals, such as sodium salicylate, is not recommended because
this can impede any further extraction and digestion procedures. However, treated gels can
be soaked in glacial acetic acid for
∼30min. and then washed several times in water to
remove fluors. For optimal recovery we prefer trying to isolate the protein of interest from
untreated gels dried onto Whatman 3MM filter paper.
Mark the sides of the gel filter with radioactive india ink and expose to film. After developing
the film, align the ink marks on the dried gel with its imprint on the film over a light box. Once
completely aligned, staple the film to the edges of the dried gel. Place this sandwich film-
side-down on the light box and trace the band using a soft pencil on the back of the Whatman
filter. Do not use a felt tip pen. For Coomassie blue stained gels, where you can easily
visualize the band of interest, you may not need to do this. Remove the staples and the film
from the dried gel and place it on a hard flat surface such as on a thick glass plate. Carefully
excise the band using a single-edge razor blade. Next peel the paper backing from the gel
slice while immobilizing it with forceps, and remove any paper fibers by scraping the back of
the slice with a razor. Try to remove as much of the paper as possible with out “shaving”
down the gel slice.
Place the gel slice in a Kontes disposable tissue grinder tube (looks like a 1.5 ml Eppendorf
tube) or a 1.7ml Sardstadt screw cap centrifuge tube. If your sample is labeled with P
32
then
determine how many cpm were recovered by counting Cherenkov radiation before
proceeding. Add about 500
μl of freshly prepared 50mM ammonium bicarbonate
(400mg/100ml), pH 7.3-7.6. The pH of freshly prepared ammonium bicarbonate is usually
within the range of 7.3-7.6. It will drift to a more alkaline pH (
∼8.3) overnight and can be used
during the enzymatic cleavage steps (see below).
Let the gel piece rehydrate for about 5min at room temperature (RT). Using the plastic
pestels, which accompany the disposable tissue grinders (Kontes), grind the gel piece until it
is fine enough to pass through a yellow Gilson pipet tip (200
μl). For multiple samples the
pestles may be inserted into a hand held, variable-speed electric drill to homogenize each gel
piece. Change, or at least wash, the pestles between the samples. Transfer the gel
suspension to a Sarstedt screw-top tube (1.5 ml). Add 500
μl ammonium bicarbonate to the
Sarstedt tube (total volume of ammonium bicarbonate solution used is 1.0ml). To the tube
containing the gel slurry, add 50
μl of β-mercaptoethanol and 10μl of 10% SDS. Tightly screw
on the lid, vortex briefly, then boil this mixture for 2-3min.