2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 105

be used, and 450 L is the volume that should be loaded when

using 24-cm IEF strips. The procedure for labeling the preparative
sample differs from the procedure for analytical gels in several
aspects and is therefore described separately:

μ

1. Prepare a protein lysate (see Notes 15 and 16) using the same

lysis procedure as used for the analytical protein amounts. The
desired amount of protein (usually 100-500 μ g) must be con-

tained in a fi nal volume of 250 μ L of lysis buffer.

2. Transfer 250 μ L of this solution to a microcentrifuge tube.

3. Calculate the amount of TCEP necessary by scaling up the

amount determined in the optimization experiment (see
Subheading 3.3 , step 3), using the formula:
amount of TCEP(nmol)

= (amount of protein to be labeled /

amount of protein used in the
optimization experiment) * optimal
amount of TCEP in optimization
experiment

(e.g., 500 μ g protein/5 μ g protein * 2 nmol TCEP = 200 nmol

TCEP).

4. Freshly dissolve the calculated amount of TCEP in 10 μ L of

water.

5. Transfer 10 μ L of the TCEP solution to the protein lysate.

6. Mix vigorously and thoroughly by pipetting, taking into

account the high viscosity of concentrated protein solutions
(see Note 17).

7. Spin down the sample in a microcentrifuge.

8. Repeat steps 6 and 7 to make sure the solution is really homo-

geneous and is located completely at the bottom of the micro-
centrifuge tube.

9. Incubate at 37°C for 60 min in the dark.

10. Calculate the required amount of Cy3 by scaling up the amount
determined in the optimization experiment (see Subheading 3.3 ,
step 3), using the formula:

amount of Cy3(nmol) = (amount of protein to be labeled /

amount of protein used in the
optimization experiment)* optimal
amount of Cy3 in optimization
experiment

(e.g., 500 μ g protein/5 μ g protein * 4 nmol Cy3 = 400 nmol

Cy3).