- Data Analysis
- Other Protocols
Bis-Tris Western Blot Protocol
A new method of western blotting utilizes a different type of gel buffer, called bis-tris buffer. This has become more popular in recent years, and we recommend it as the absolute best protocol for standard western blot techniques.
This method is very similar to the standard method, with two major differences. First, the stacking and resolving gels are buffered with bis-tris instead of tris-HCl. Second, the running buffer is MOPS-SDS running buffer instead of tris-glycine-SDS running buffer. Everything else, including lysis, loading, transfer, blocking and development, is the same.
You will notice that the gel buffer replaces tris-HCl with bis-tris-HCl and excludes SDS from the gel. To compensate for the lack of SDS, a reducing agent called Sodium Bisulfite is included in the MOPS-SDS running buffer to prevent re-oxidation of proteins while the gel is running.
Bis-Tris Western Blotting Reagents
There are some buffers that are unique to the Bis-Tris western blot protocol. They are listed below. Again, use the molarity calculator from GraphPad if you need help calculating the buffer recipes.
Bis-Tris HCl Gel Buffer (3.5x Stock)
- 1.25 M Bis-Tris HCl pH 6.8
Use this buffer for both the stacking and resolving gels to carry out the Bis-Tris western protocol.
MOPS-SDS Running Buffer (5x Running Buffer)
- 250 mM Tris
- 250 mM MOPS
- 5 mM EDTA
- .5% SDS
- 5 mM Sodium Bisulfite (Add fresh before run from a 1M stock)
To make 2 L of 5x running buffer:
- 60.57 g Tris
- 104.63 g MOPS
- 20 ml of .5M EDTA
- 50 ml of 20% SDS
Protocol for Bis-Tris Western Blots
Bis-Tris gels should be made according to the gel recipes. As discussed on the gel casting page, it is best if the gels are made the night before the western blot is to be run. Sample preparation for bis-tris western blots is identical to sample preparation for standard western blots.
- Running buffer should be made by diluting the stock MOPS-SDS running buffer to 1x. We purchase 20x MOPS-SDS running buffer from a commercial vendor, but we’ve also provided you with a recipe for 5x buffer if you choose to make it in your own lab. For most western blot apparatuses, 500 ml is a sufficient quantity of running buffer.
- After diluting the running buffer, add fresh sodium bisulfite to it. This will act as a reducing agent during running . The sodium bisulfite may have a very bad odor, so be sure to use it under a hood. We use a 1M stock of sodium bisulfite and add 2.5 ml to 500 ml 1x MOPS-SDS running buffer. It is best if you include the sodium bisfulfite in your dilution of the 5x MOPS-SDS buffer so that the final concentration is 1x and the total volume is exactyl 500 ml. Alternatively, you can add 250 mg of sodium metabisulfite poweder to your 500 ml of 1x MOPS-SDS running buffer.
- Load and run your gels as you do with standard western blots. It helps to load any empty lanes with Laemlli buffer to keep the gel running straight. Run the gel at 100V for approximately 2 hours. The time will vary depending on gel percentage and desired resolution.
- People have reported elsewhere, and we have seen it in lab, that the bis-tris gels may change pH at the dye front. This is often accompanied by a slight abnormality or deformity in the gel lanes at the very bottom of the gel. I do not have this problem, but a summer student of mine struggled with it. It helps to run the gel on ice, or at 4°C in the cold room. We have not had this problem when running the western blot gel at cold temperatures.
- After you have the desired resolution, stop the run, take the gels out, and place them into standard transfer buffer. For bis-tris western blots, you’ll need to let the gel equilibrate for 10 minutes in the transfer buffer. So, after the gel is cut out and placed in transfer buffer, you can get the membranes and filter paper ready while the gel is equilibrating.
- Finally, load the semi-dry transfer apparatus as you normally would: From top to bottom, 3 blotting papers, membrane, gel, 3 blotting papers.
- Transfer at 15V for 45 minutes.
The rest of the bis-tris western blot protocol is identical to the stand western blot protocol. We find that the bis-tris western blots give sharper bands that never overlap. They are incredibly reproducible and very useful for western blot quantification by densitometry because the lanes are so distinct.