Proteomic Analysis of Redox-Dependent Changes Using Cysteine-Labeling 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 126

5. Incubate with the appropriate primary antibody for 2 h at
room temperature or overnight at 4°C on a rocking platform.
6. Remove the primary antibody and wash the membrane 3 times
for 15 min in TBS-T on a rocking platform.
7. Incubate with the appropriate HRP-conjugated secondary
antibody for 1 h at room temperature on a rocking platform.
8. Remove the secondary antibody and wash the membrane 3
times for 15 min in TBS-T on a rocking platform.
9. Mix the two ECL reagents 1:1 and incubate immediately with
the membrane for 1-2 min ensuring full coverage of the
membrane.
10. Remove the membrane from the ECL reagents, drain excess
fl uid, enclose the membrane in Saran wrap, and tape it into an
X-ray cassette.
11. In a darkroom, place an X-ray fi lm on the top of the membrane
and close the cassette. Leave to expose for an appropriate
period to give a reasonable signal that is not saturated.
12. Scan the X-ray fi lm on a Bio-Rad GS-800 densitometer and
quantify the intensity with QuantityOne software.
4. Notes
1. The synthesis and purifi cation of the ICy dyes are detailed in ( 13 ).
2. Urea decomposes to ammonium cyanate at temperatures above
30°C. The ammonium cyanate subsequently modifi es the pri-
mary amino groups of proteins via carbamylation resulting in
altered p I .
3. The lysis buffer should not contain thiourea, which competes
with the ICy dyes. Thiourea is often used in 2-DE sample
buffers to improve protein solubility.
4. Prepare the staining solution at least 1 h prior to use, consider-
ing that it takes the ammonium sulfate a while to dissolve
completely.
5. It is recommended that at least four replicate assays are per-
formed for each sample for accurate protein determination.
Dilute concentrated samples with lysis buffer if necessary.
6. For ICy labeling, cells are lysed in the presence of dye to limit
post-lysis thiol modifi cation. Since ICy dyes interfere with the
protein assay, protein concentrations are determined on replica
lysates not containing the ICy dyes.