Quantcast
Skip to content

Laemmli Buffer: What Is It for Anyway?

Caricamento di un gel di agarosio

Electrophoresis encompasses a wide range of techniques in which charged biomolecules in a liquid, a solid, or a semisolid solution can be separated by size under the application of an electric field. The most common application of electrophoresis for the separation of proteins is SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), which has been previously covered here 

The most important step of electrophoresis is actually the extraction of proteins from a sample. During this process certain conditions need to be met in order to get an optimal result. Although protein extraction methods can vary, Laemmli buffer is a constant in nearly all protocols.
 

Taking its name from Prof. Ulrich K. Laemmli who refined the procedure of SDS-PAGE in the 1970s, Laemmli buffer creates excellent conditions for quality separation of proteins based on their size. Laemmli buffer contains: (1) sodium dodecyl sulfate (SDS); (2) a thiol agent; (3) glycerol; (4) tris-hydroxymethyl-aminomethane (tris); and (5) a color agent, like bromophenol blue. In order for someone to be able to master the technique of SDS-PAGE it is useful to know the reason and chemistry behind all of these reagents. 

The Line-Up 

1. SDS  

Well-known as a detergent, SDS denaturates proteins by disrupting non-covalent linkages and destabilizing natural conformations. By acting as an anionic surfactant, it imparts net negative charge on proteins, causing repulsion between amino acids, which leads to protein unfolding and a somewhat trend towards linearization.  It is estimated that 1.4 gram of SDS can bind 1 gram of protein.  

The same principal is taken into consideration when SDS is used in hygiene industry and more specifically as detergent to successfully remove stains. The amphipathic nature of SDS, along with its denaturation function, allows the detergent to easily remove proteins or fatty acids of a stain. 

2. Thiol agent 

Thiol agents, such as beta-mercaeptoethanol or dithiothreitol (DTT), are used to reduce the disulphide bonds created between sulphur containing amino acids like cysteines. Also, because thiol agents have antioxidant properties, they are able to prevent oxidation of cysteines. Thus, thiol agents work with SDS for successful protein linearization.  

3. Glycerol 

Once your samples have been successfully linearized, it is time to get them set up in the wells of the gel you are using for electrophoresis. To ensure that your protein sample(s) won’t float away, high-density glycerol is included in Laemmli buffer, essentially weighing your sample down so it stays in its designated well. 

4. Tris 

During SDS-PAGE, Tris is being used to control three different pH levelsHowever, when it is a part of the Laemmli buffer, Tris functions to maintain pH 6.8 to stabilize your protein extract for several days at the fridge, without compromising your proteins. At the same time, Tris can inhibit enzymatic reactions and prevent cell proteases from degrading your proteins of interest. 

5. Colour agent 

Finally a colour agent (or dye) like bromophenol blue is useful for visualizing your sample in the well and tracking its progress through the gel later.  

How to Use Laemmli Buffer 

For your notebook, a common and easy to make recipe for a 2X concentrated Laemmli buffer is: 4% SDS, 10% beta-mercaeptoethanol, 20% glycerol, 0.1? Tris pH 6.8, and 0.005% of bromophenol blue.  

A concentrated Laemmli buffer can be stored at 4oC for at least a year without worrying about its effectiveness. It is always a good idea to make a 2X or 5X stock and dilute it during your protein extraction.  

So now you have the know-how in order prepare your proteins for successful electrophoresis, but also how detergents are working inside your washing machine. Two birds with one stone!
 

References  

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature, 227(5259), 680. 

Leave a Comment





This site uses Akismet to reduce spam. Learn how your comment data is processed.

Scroll To Top
Share via
Copy link