Laemmli buffer

From Molecular Biology Wiki

History of the Laemmli buffer

Laemmli buffer was first created by Laemmli in 1970. ^[1]

Laemmli buffer Background

Laemmli sample buffer is especially formulated for protein sample preparation to be used in the Laemmli SDS-PAGE system. 2X sample buffer is added to each protein sample at a 1:1 ratio, and is boiled (or heated) on a heating block for 1-5 min.

Purpose of the Laemmli buffer

The beta 2-mercaptoethanol reduces intra and inter-molecular disulfide bonds of the proteins to allow proper separation not by shape but by size.

The SDS detergent binds to all the proteins positive charges which occur at a regular interval, thus giving each protein the same overall negative charge so that proteins will separate based on size and not by charge. The SDS also denatures the proteins and subunits to also help separate them based on size, not on shape. SDS binds to proteins at about 1.3g of SDS / g of protein.

Bromophenol blue serves as an indicator dye, and migration indicator where one can observe the dye front that runs ahead of the proteins. Bromophenol blue also functions to make it easier to see the sample during loading of the gel wells with protein sample. Glycerol in the Laemmli buffer increases the density of the sample so that it will fall to the bottom of the well, minimizing puffing or loss of protein sample in the buffer, and layer in the sample well.

DTT is present in many formulation to help reduce any disulphide S-S bonds that could provide secondary/tertiary structure and/or dimer formation.

Laemmli Buffer Recipe Preparation

Basic 2X Laemmli Buffer contains:

• 4% SDS
• 20% glycerol
• 10% 2-mercaptoethanol
• 0.004% bromphenol blue
• 0.125 M Tris HCl

The solution has a pH of approximately 6.8.

Other formulations have been devised including:

Sample Buffer (4X Laemmli Buffer)

• 2.4 ml 1 M Tris pH 6.8 (Same as upper gel buffer)
• 0.8 g SDS stock
• 4 ml 100% glycerol
• 0.01% bromophenol blue. Final Concentration is .02%
• 1 ml ß-mercaptoethanol (electrophoresis grade)
• 2.8 ml water

Recommended Storage Temperature of Laemmli buffer

Storage temperature should be at −20°C.

Laemmli buffer References

1. ↑ Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, (1970) Abstract