Western blotting is the most common assay to measure protein levels from cells and tissues. Standard western blots start with soluble proteins in a detergent-containing buffer. Normally, these proteins will be denatured by boiling and reducing agents (e.g. Beta-mercaptoethanol). Bis-tris westerns begin the same way, and, in fact, are completely identical to standard western blots, with a few major exceptions.
First, the buffer used to make the gels is different. As you can probably guess from its name, bis-tris gels use bis-tris-HCl buffer, whereas traditional western blots use standard tris-HCl buffer. The second difference is that the stacking gel and resolving gel use the same buffer during bis-tris western blotting. In standard applications, the stacking gel is acidic (pH 6.8) and the resolving gel is basic (pH 8.8). For bis-tris westerns, the entire gel is run under acidic conditions at pH 6.8.
First, the buffer used to make the gels is different. As you can probably guess from its name, bis-tris gels use bis-tris-HCl buffer, whereas traditional western blots use standard tris-HCl buffer. The second difference is that the stacking gel and resolving gel use the same buffer during bis-tris western blotting. In standard applications, the stacking gel is acidic (pH 6.8) and the resolving gel is basic (pH 8.8). For bis-tris westerns, the entire gel is run under acidic conditions at pH 6.8.
The acidic conditions of bis-tris westerns favor the reoxidation of proteins during electrophoresis. To compensate for this problem, a reducing agent (Sodium Bisulfite) is added to the running buffer at 2.5 mM concentration. Finally, the last major difference is the constitution of the running buffer. For bis-tris westerns, use a MOPS-SDS running buffer, in contrast to the traditional tris-glycine-SDS running buffer of standard western blots.
A general breakdown of the bis-western protocol will be as follows:
Cast your gels with bis-tris HCl pH 6.8 buffer at the desired acrylamide concetration (6-15%). It is best if you do this the night before you intend to run your gel. The best results are obtained when you allow the gel to polymerize overnight at 4 degrees Celsius.
Boil and denature your samples.
Make up MOPS-SDS running buffer containing 2.5 mM sodium bisulfite. Add the bisulfite fresh before the run.
Run the gel at a constant voltage of 100V for approximately two hours. The run-time will vary depending on the concentration of the gel.
Once the desired resolution is achieved, transfer the gel to a membrane using the traditional tris-glycine-SDS transfer buffer with 20% methanol.
Let the gel sit in transfer buffer for 10 minutes prior to semi-dry transfer so that the MOPS-buffered gel equilibrates with the transfer buffer.
Transfer at 15V for 45 minutes for 1 mm thick gels.
Once the transfer is complete, block the membrane with either 5% bovine serum albumin in tris-buffered solution with tween-20 (TBST) or 5% milk in TBST for two hours.
Incubate with your primary antibody for four hours to overnight
Wash the primary antibody off and incubate with horseradish peroxidase-conjugated secondary antibody for one hour.
Wash the secondary antibody off, immerse the western blot in Enhanced Chemiluminescence reagents, and expose it to film.
Bis-tris westerns are very reliable and reproducible. Consequently, they save you quite a bit of frustration, time, and productivity due to their superior consistency compared with regular western blots.
Article Source: Joe Yossarian
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