Bio-Rad Criterion™ TBE-Urea Precast Gels - Manual (Page 43)

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To visualize total protein on blots using the Gel Doc EZ imager, refer to Section 5.4.
13.4 Immunodetection
After transfer, blots are ready for downstream processing. While all protein and antibody combinations
are different and may require optimization, a general protocol for the immunodetection of a large
number of protein and antibody combinations is provided (see Appendix B for buffer formulations).
1. Immediately after transfer, place the membrane into Tris-buffered saline with Tween 20 (TTBS)
containing blocking agent (for example, 3% BSA, 5% nonfat dry milk, 1% casein, or 1% gelatin).
Incubate at room temperature with agitation for 1 hr.
2. Dilute the primary antibody in blocking solution (suggested dilution is specified by the manufacturer).
Incubate the blot in the primary antibody solution at room temperature and with agitation for 1 hr.
3. Wash the blot with TTBS as directed in the instructions for the detection method to be used (for
example, 5 times, 5 min each at room temperature).
4. Dilute the secondary antibody into TTBS as specified by the manufacturer. Incubate the blot in the
secondary antibody solution at room temperature with agitation for 1 hr.
5. Wash the blot with TTBS for 5 min at room temperature with agitation. Pour off the wash solution
and repeat 5 times.
6. Follow the directions for the detection kit used to develop the blot. For the Immun-Star ™ WesternC ™
chemiluminescence kit (catalog #170-5070), mix 3 ml luminol/enhancer with 3 ml peroxide solution
to make a 1x working solution for a 7 x 8.5 cm membrane. Incubate the membrane in the solution
for 3–5 min. Prior to imaging, drain the excess substrate and place the membrane in a protective
sleeve (such as plastic wrap) to prevent drying.
Instruction Manual and Application Guide