Standard Western Protocol Buffers

Below is a list of the buffers required to carry out a western blot. We will provide you with the final concentrations. If you need help calculating molarity and required mass to make solutions, we suggest you use GraphPad’s molarity calculator.

Laemmli Buffer (1x)

• 60 mM Tris HCl pH 6.8
• 2% SDS
• 0.02% Bromophenol Blue
• 5% β-Mercaptoethanol

Tris-Glycine-SDS Running Buffer

• 25 mM Tris pH 8.3
• 200 mM Glycine
• 0.1% SDS
• We recommend buying 20x running buffer commercially, as it is fairly inexpensive.

1.5M Tris HCl pH 8.8

•  To make 500 ml:
• Dissolve 90.8 g tris base in 400 ml ultrapure water.
• pH to 8.8 with 37% HCl.
• Bring volume to 500 ml.

1.0M Tris HCl pH 6.8

• To make 500 ml:
• Dissolve 60.5 g tris base in 400 ml ultrapure water.
• pH to 6.8 with 37% HCl.
• Bring volume to 500 ml.

Transfer Buffer (For a semi-dry transfer apparatus)

• 20% Methanol
• 50 mM Tris
• 40 mM Glycine
• .0375% SDS
• pH should be approxinately 9.0 (It shouldn’t have to be adjusted)

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