The Laemmli sample buffer / Laemmli buffer is used for the better isolation of proteins in SDS-PAGE gel electrophoresis. The buffer is connected with the invention of SDS-PAGE during the quest for finding T4 phase proteins and got its name after the inventor Prof. Ulrich K. Laemmli [1]. The composition has been discussed since the 70s and alternatives have been proposed. Nevertheless, the Laemmli-based solution is still used and sold by companies with minor differences.
Commonly used alternatives are
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Phosphate modification of the Laemmli is known to reduce unexpected protein cleavage, thanks to the better-buffering capacity of phosphate at used pH [2].
pH 6.8 is used, although it is below the buffering capacity of Tris (pH 7-9). The nearly neutral pH is used because low pH causes the peptide bonds to hydrolyse and on the other hand, high pH disrupts the activity of thiols.
The pH is normally adjusted with HCl and NaOH.
The Laemmli buffer is often prepared as a 2X or 4X solution and is mixed with the sample to 1X.
Standard Laemmli sample buffer contains:
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1Tris base is tris (hydroxymethyl) aminomethane. You can avoid using crystalline Tris by using Tris buffer, adjusted with HCl to 6.8.
2SDS is sodium dodecyl sulfate.
3Bromphenol blue is available as sodium salt or solution. Some modern protocols are using higher concentration (0.005 % Bio-rad, 0.002 % Sigma-Aldrich) to obtain bright colour.
Tris as a buffering substance. As discussed above, the tris buffering system and the pH play an essential role in preserving peptide bonds from breaking apart.
SDS: Proteins comes in different sizes and charges. SDS helps in linearizing (by denaturing) the proteins and bringing a net negative charge to the proteins irrespective of the initial charge. This minimizes variations in the variations in the movement of proteins in the gel otherwise skewed by the difference in charge and shape.
Glycerol: The high density (thickening of the solution) of glycerol ensures the sample moves down into the well.
beta-mercaptoethanol: is used for breaking the disulphide bonds. beta-mercaptoethanol, along with SDS, ensure the bands are due to individual polypeptides instead of molecular complexes.
Bromophenol blue: visually indicates the location (tracking dye) of the sample in the gel.
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Although the reagents mentioned above are used in standard Laemmli buffer, variations of the buffer have other substitutes. The following table represents which reagent in the buffer is substituted with others.
Procedure (50 ml):
In the next step, you have two options for adding beta-mercaptoethanol:
Option 1:
Option 2:
Option 1 is for quick use, choose the second option for better results.
If any thiol was added, store at -20 °C or use quickly.
Without thiols, it can be stored at room temperature or at 4°C.
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