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.
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.