Laemmli buffer: Preparation (1x,2x & 4x) and principle

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    Introduction

    The Laemmle sample 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

    • Morris SDS-PAGE sample buffer (very similar composition) or
    • Phosphate modification of Laemmli sample buffer

    Phosphate modification of the Laemmle is known to reduce unexpected protein cleavage, thanks to the better-buffering capacity of phosphate at used pH [2].

    Uses

    • This buffer is used as a sample preparation buffer for protein samples in SDS-PAGE, compatible with Tris-glycine-SDS running buffer.
    • It can be used for resuspension of Immunoprecipitation beads before SDS-PAGE.

    pH

    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.

    Composition

    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:

    ReagentMolecular weightConcentration (M or %)Add for 50 ml of 2XAdd for 50 ml of 4X
    1X2X4X
    Tris base1121.140.0625 M0.125 M0.250 M0.747 g1.514 g
    SDS2288.370.07 M (2%)0.14 M (4%)0.28 M (8%)2 g4 g
    glycerol92.0910%20%40%10 ml20 ml
    2-mercapto-ethanol78.135%10%20%5 ml10 ml
    Bromphenol blue3691.94---1 mg2 mg

    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.

    Note: There are many recipes available. We have provided some of them at the end. They may be used as an alternate to the composition given above.

    Reagents

    • Tris base
    • SDS
    • glycerol
    • 2-mercaptoethanol
    • bromophenol blue or the alternatives mentioned above.
    • HCl (concentrated), NaOH for adjustment

    Role of reagents 

    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.

    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.

    Instruments and other requirements

    • Glass beaker
    • Weighing balance
    • Magnetic stirrer and pellet
    • pH meter
    • Measuring cylinders
    • Pipette (glass or automatic with tips)

    Procedure

    Procedure (50 ml):

    1. Prepare the Tris solution by dissolving the exact amount of Tris base in 10 ml of water in a beaker. Use magnetic stirrer if needed.
    2. Adjust the pH to 6.8 with concentrated HCl. Be careful not to overshoot.
    3. Add glycerol to the tris solution using a cylinder and mix well.
    4. Add the exact amount of SDS and bromphenol blue. It takes time to dissolve, stirrer recommended.

    In the next step, you have two options for adding beta-mercaptoethanol:

    Option 1:

    1. Add mercaptoethanol and adjust the total volume to 50 ml.
    2. Divide in aliquots.
    3. store at -20 °C.

    Option 2:

    1. Adjust the volume so you can add mercaptoethanol just before use (e. g. for 2X adjust to about 45 ml).
    2.  Divide in aliquots and store.
    3.  Add mercaptoethanol just before use.

    Option 1 is for quick use, choose the second option for better results.

    Storage

    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.

    Caution

    • Concentrated HCl fumes are dangerous, use it in a fumigation hood.
    • Mercaptoethanol is seriously irritating and toxic if swallowed or inhaled, so it is advised to handle it in an active fumigation hood.
    • Tris base is a skin and eye irritant as well as bromphenol blue.

    Reference

    1. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4.
    2. Modification of the Laemmli Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Procedure to Eliminate Artifacts on Reducing and Nonreducing Gels.
    3. Sigma-Aldrich.
    4. Bio-rad.

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