Biology Reference
In-Depth Information
2. Add cell lysis solution or sodium hydroxide (1 M) to adjust
the pH of acidic samples to around pH 8.5 (see Note 9).
3. Recalculate the protein concentration of the pH-adjusted
sample, if required.
1. Transfer a small volume of DMF with a syringe and needle into
a microfuge tube.
2. Take the vial of CyDye™ DIGE fl uors from a −20°C freezer
and leave it to warm up at room temperature for 5 min.
3. Reconstitute CyDye™ DIGE fl uors with DMF to give a stock
concentration of 1 nmol/mL. For example, add 25 mL of DMF
to 25 nmol of fl uor.
4. Replace the cap of the microfuge tube containing the dye and
vortex vigorously.
5. Centrifuge briefl y to bring the reconstituted CyDye™ contents
down to the bottom of the tube. Store the remaining stock of
CyDye™ at −20°C and protect from light as soon as possible.
6. Add 1 volume of CyDye™ stock solution to 1.5 volumes of DMF
to make a diluted CyDye™ working solution (400 pmol/mL).
3.5.2. Reconstitution
of CyDye™
1. Prepare a 1-mm thick, 12.5% SDS-PAGE gel by mixing 1.6 mL
of 40% acrylamide/bis solution with 1.25 mL of 1.5 M Tris-
HCl pH 8.8 separating buffer, 50 mL of 10% SDS, 50 mL of
10% APS, 2.05 mL of water, and 2.5 mL of TEMED. Pour the
gel solution into the mini-PROTEAN 3 gel-casting assembly
and leave suffi cient space for the stacking gel. Overlay with
200 mL of water-saturated butanol. Allow the gel to polymerize
for at least 30 min.
2. Pour off the water-saturated butanol.
3. Prepare the stacking gel by mixing 200 mL of 40% acrylamide/
bis solution with 252 mL of 1 M Tris-HCl-stacking buffer pH
6.8, 20 mL of 10% SDS, 20 mL of 10% APS, 1.51 mL of water,
and 2 mL of TEMED.
4. Pipette in the stacking gel and insert the comb. Allow the gel
to polymerize for 30 min.
5. Prepare the running buffer by diluting 100 mL of 10× running
buffer with 900 mL of ultrapure cold water. Mix the content
and leave the running buffer at 4°C.
6. Transfer 5 mg of sample into a microfuge tube.
7. Add 0.1 mL of diluted CyDye™ working solution to the tube
(i.e., 5 mg of protein is labeled with 40 pmol of CyDye™).
8. Mix and centrifuge briefl y. Leave on ice for 30 min in the dark.
9. Add 0.1 mL of 10 mM lysine to quench the reaction. Mix and
centrifuge briefl y.
3.5.3. Labeling a Small
Amount of Protein to Test
the Labeling Effi ciency
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