Biology Reference
In-Depth Information
9. Centrifuge the CM at 2,000 ×
g
at 4°C for 10 min to remove
cell debris, and transfer the supernatant to a new tube.
10. Add protease inhibitor tablets to the CM according to the
manufacturer's instructions.
11. Filter the CM through a 0.1-
μ
m membrane fi lter and collect
the fi ltrate in a new tube.
12. Concentrate the filtrate to a final volume of 1 mL using
centrifugal fi ltering devices (see Note 4).
13. Precipitate proteins using the 2D Clean-Up Kit according to
the manufacturer's instructions (see Note 5).
14. Re-solubilize secretome proteins in 2DE sample buffer.
15. Determine protein concentration using the 2D Quant Kit,
following the manufacturer's instructions (see Note 6).
16. Snap freeze the purifi ed secretome sample and store at −80°C,
or proceed with protein labeling.
1. Prepare the stock dye solutions (1 nmol/
L) by reconstituting
25 nmol of Cy2, Cy3, and Cy5 dyes each with 25
μ
3.2. CyDye
Reconstitution
and Protein Labeling
μ
L of DMF
(see Note 7).
2. Vortex vigorously to dissolve the dye, which should produce
deep colors of Cy2-yellow, Cy3-red, and Cy5-blue.
3. Prepare the working dye solution (200 pmol/
μ
L) by diluting
L of DMF.
4. Store the remaining stock Cy dye solutions in a light-excluding
container at −20°C (see Note 8).
5. Ensure that each secretome sample is between pH 8.0 and 9.0
prior to protein labeling, and adjust as necessary.
Each secretome sample is labeled twice with Cy3 and twice
with Cy5 to eliminate dye specifi c bias, and separated
across six 2D gels. The internal standard labeled with Cy2
is generated by combining all secretome samples, and run
on every gel to coordinate inter-gel spot matching and
reduce gel-to-gel variation (see Note 9).
6. Label 25
1.5
μ
L of each stock dye solution with 6
μ
g of each secretome sample (MDCK (a), MDCK
(c), 21D1 (b), 21D1 (d), 21D1 + TFG-
μ
β
(a), 21D1 + TFG-
β
(c)) with 1
μ
L of Cy3 working dye solution.
7. Label 25
g of each secretome sample (MDCK (b), MDCK
(d), 21D1 (a), 21D1 (c), 21D1 + TFG-
μ
β
(b), 21D1 + TFG-
β
L of Cy5 working dye solution.
8. Combine 12.5
(d)) with 1
μ
μ
g from each of the 12 secretome samples, and
L of Cy2 working dye solution (see Note 10).
9. Store the remaining working Cy dye solutions in a light-
excluding container at −20°C (see Note 11).
label with 6
μ
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