Analysis of Serum Proteins by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 285

and 4 L TEMED. Directly after addition of TEMED

pour it on top of the separation gel and insert the comb
until polymerized (

μ

∼

30 min).
4. In the meantime prepare the sample by mixing 5-10 gof

depleted fraction with 5X sample buffer (max. loading on
a 0.75 mm thick gel is 25

μ

L). Heat samples at 95 ◦ Cfor
15 min, then cool on ice, and briefly centrifuge (

μ

∼ 15 s).

The samples are ready for loading.
5. When the stacking gel is polymerized, remove the comb
and place the gel into the running chamber. Fill the upper
and lower gel chamber with 1X running buffer (10X
diluted with MilliQ water) and wash the slots with 1X run-
ning buffer using a 1mL syringe with a 25 gauge needle
attached.
6. Load the marker (5

L PageRuler Prestained Protein Lad-
der) and the samples on the gel. Run the gel at 20 mA
through the stacking gel and 25 mA afterwards, until the
blue loading dye leaves the separation gel.
7. Stop the run and disassemble the gel unit. Discard the
stacking gel and transfer the separation gel carefully into
a clean container.
8. Silver staining, using the Silver-Stain Plus Kit, is undertaken
according to the Bio-Rad Protocol. The gel is fixed in fix-
ative solution (50% methanol, 10% acetic acid, 10% fixative
enhancer, 30% MilliQ water) for 20 min.
9. Rinse the gel several times with MilliQ water, incubating
for 10 min each time before replacing with fresh MilliQ
water.
10. To stain and develop the gel with developer solution, dis-
solve 0.5 g development accelerator solution in 10 mL
MilliQ Water. Then mix in the following order 7 mL
MilliQ water, 1 mL silver complex solution, 1 mL reduc-
tion moderator solution, and 1 mL image development
reagent. Combine this mix with 10 mL development accel-
erator solution and immediately apply to the gel after dis-
carding the MilliQ water.
11. When the desired staining is reached, stop the reaction by
incubating the gel in 5 % acetic acid solution. The protein
pattern from the depleted samples should be similar, but
should differ from a normal serum sample containing the
highly abundant proteins ( see Fig. 17.2 ) .

μ

μ 3.7.Digestion

withTrypsin

1. Add SDS to 200-400 g of depleted serum in a 1.5 mL

microfuge tube to a final concentration of 0.07% using a 2%
stock solution (prepared in water and filter sterilized).
2. Add TCEP to a final concentration of 1.78 mM.