Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis - Manual (Page 34)

11.3 Total Protein Blot Stains
Total protein staining of a membrane provides an image of the complete protein pattern, which is
required for the full characterization of specific antigens detected in complex protein mixtures. Gels
shrink during staining, so comparison of an immunologically probed membrane to a stained gel is
not practical. Instead, the exact location of a specifc antigen is determined by comparing two blotted
membranes: one that has been probed with an antibody and the other stained for total protein.
Method
Sensitivity Protein
Load
(μg/Band)
Advantages
Disadvantages
Imaging
SYPRO Ruby
protein blot stain 2–8 ng ~0.2 Compatible with
mass spectrometry,
Edman-based
sequencing,
and standard
immunological
procedures Multistep protocol
requires UV, LED,
or laser imaging for
maximum sensitivity Fluorescence
visualization
with UV, LED
epi-illumination or
laser scanning
Colloidal gold stain 1 ng ~0.1 High sensitivity;
single-step protocol Incompatible with nylon
membranes Photography with
epi-illumination
or reflectance
densitometry
Anionic dyes
(amido black,
Coomassie R-250,
Ponceau S, Fast
Green FCF) 100–1,000 ng ~5.0 Inexpensive, rapid Low sensitivity
Table 11.3. Total protein blot stains.
11.4 Immunodetection
After transfer, blots are ready for downstream processing. Though all protein and antibody combinations
are different and may require optimization, a general protocol for immunodetection of a large number of
protein and antibody combinations is listed below. 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) and
incubate either for 1 hr at room temperature or overnight at 4°C.
2. Dilute the primary antibody in blocking solution (dilution is specified by the manufacturer). Incubate
at room temperature with agitation for 1 hr.
3. Wash the blot with TTBS as directed (for example, five times, 5 min each at room temperature with
agitation).
4. Dilute the secondary antibody into TTBS as specified by the manufacturer. Incubate the blot in the
secondary solution at room temperature with agitation for 1 hr.
5. Wash the blot with TTBS five times, 5 min each at room temperature with agitation.
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.
To visualize total protein on blots using the stain-free system, see Section 5.4.
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Mini-PROTEAN Precast Gels