Western Blot Protocol – Buffers

Running, Transfer, and Blocking buffers

Laemmli 2X buffer / loading buffer
4% SDS
10% 2-mercaptoethanol
20% glycerol
0.004% bromophenol blue
0.125 M Tris-HCl
Check the pH and adjust pH to 6.8.

Running buffer (Tris-Glycine/SDS)
25 mM Tris base
190 mM glycine
0.1% SDS
Check the pH, which should be about pH 8.3. Adjust if necessary.

Transfer buffer (Wet)
25 mM Tris base
190 mM glycine
20% methanol
Check the pH, which should be about pH 8.3. Adjust if necessary.
For proteins larger than 80 kDa, we recommend that SDS is included at a final concentration of 0.1%.

Transfer buffer (Semi-dry)
48 mM Tris
39 mM glycine
20% methanol
0.04% SDS

Blocking buffer:
5% milk or BSA (bovine serum albumin)
Add to TBST buffer. Mix well and filter. Failure to filter can lead to “spotting” where tiny dark grains will contaminate the blot during color development.

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