Download Laemmli Buffer Recipe Preparation

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
4x SDS Protein Sample Buffer
SDS protein sample buffer is added to solubilized protein samples prior to analysis by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). 1 part of this 4x buffer is added to 3 parts solubilized protein. Alternatively, the buffer can
be diluted to 1x (with water) and added directly to pellets of bacterial or eukaryotic cells. In either case, the mixture
should be heated at 95-100 C for 5 minutes to denature proteins prior to SDS-PAGE.
Final concentrations:
40% Glycerol
240 mM Tris/HCl pH 6.8
8% SDS
0.04% bromophenol blue
5% beta-mercaptoethanol
For 10 ml:
4 ml 100% glycerol
2.4 ml 1M Tris/HCl pH 6.8
0.8 g SDS
4 mg bromophenol blue
0.5 ml beta-mercaptoethanol
3.1 ml H2O