Tris Buffered Saline (TBS) 10X recipe

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    Introduction

    Tris-buffered saline (TBS) is isotonic and non-toxic. TBS provides optimal conditions for antigen-antibody interactions. TBS composed of Tris and NaCl. While the former component gives the buffering capability, the latter one helps in tonicity (isotonic or hypertonic relative to cells depends on the NaCl concentration). There are variants in TBS which differs in the concentration of Tris (10 to 100 mM) and NaCl (150 to 500 mM). Although there are many variations in TBS, a commonly used “standard” formulation for 1X TBS contains 0.05 M Tris and 0.15 M sodium chloride, pH 7.6, at 25 °C.

    Uses

    TBS is used as washing buffer in the following applications

    • Antibody Diluent.
    • Wash buffer in immunoassays.
    • Washing buffer for alkaline phosphatase or peroxidase conjugates in dot blot assay.
    • Washing buffer for electrodes.
    • Immuno-histochemical staining.
    • In Situ hybridization.

    pH

    The pH of the medium to be adjusted to 7.4 to 8.0 depending on the application. Use HCl and NaOH for adjusting pH. TBS with pH 7.6 is used in most of the applications.

    Composition

    There are many recipes available for making TBS buffer. The following table is based on the CSHL protocol (standard) and Abcam website. For finding the links to these pages follow the links given in the reference section at the bottom of the page.

    There are two different recipes available for preparing TBS buffer which has same tris and NaCl concentration.

    Method 1

    ReagentM.WMolarity of 1X bufferMolarity of 10X bufferRecipe for 10X buffer 1L
    Tris-HCl157.6~15.2 mM~152.2 mM24 g
    Tris base121.1~4.62 mM~46.2 mM5.6 g
    NaCl58.4150 mM1500 mM88 g

    The pH of the buffer should be close to 7.6. In the case of pH adjustment, using concentrated NaOH and HCl.

    Method 2

    ReagentM.WMolarity of 1X bufferMolarity of 10X bufferRecipe for 10X buffer 1L
    Tris base121.1~20 mM~200 mM24 g
    NaCl58.44150 mM1500 mM88 g

    Once the reagents are added as per requirement, use 12N HCl for adjusting pH to 7.6

    Reagents

    • Tris base and/or Tris HCl
    • NaCl
    • HCl
    • ddH2O

    Materials and Instruments

    • pH meter
    • Magnetic stirrer and bead
    • Glass Beakers
    • Graduated cylinder
    • Paper boats

    Procedure

    preparation depends on whether you have selected Method 1 or Method 2.

    Method 1 allows you to avoid using HCl, which could be convenient when you do not have accessibility to fumehood.

    The following preparation is based on Method 1 (Table 1 in the composition section) for the preparation of 1L of 10X TBS buffer.

    1. Weigh 24 g of Tris-HCl, 5.6 g of Tris base and 88 g of NaCl.
    2. Add 900 ml of distilled water.
    3. Stir the mixture using magnetic stirrer until salts are dissolved. 
    4. Check for the pH of the solution.
    5. Adjust the pH if necessary, using concentrated HCl and NaOH.
    6. Once you are satisfied with the pH, make up the volume to 1L using distilled water. 

    Sterilization can be performed by filtration or autoclaving. Filter the buffer solution into a sterile flask through a 0.22 µm filter.

    Diluting 10X TBS to 1X

    It is a general practice in many laboratories to make stock solutions and diluting them as per requirements.

    If you have a 10X stock, mix 100 ml of stock to 900 ml of distilled water to get 1L of 1X TBS solution. Adjust the pH of the 1X if required.

    Tris-buffered saline with Tween 20 (TBS-T)

    The first use is probably connected with the invention of Western blot, where the blot was washed in Tris-buffered saline with NP 40 as a detergent [1,2]. Since then, the composition remained nearly the same, but NP 40 was replaced by Tween 20 by most of the authors.

    PBST can replace TBST in most of the usages. The most significant difference is in buffering substance – PBST uses phosphate instead of Tris. The phosphate shows interference with alkaline phosphatase-conjugated antibodies and rises background signal utilizing phospho-specific antibodies (GE; Termofisher).

    To sum it up, it is TBS-T is TBS with Tween 20 (0.05-0.1 %). Lower concentration of Tween is recommended for weakly binding antibodies (GE).

    Storage

    Store the solution at room temperature or at 4oC. Depending on the source the TBS buffer can be stored a minimum of 3 months to 2 years.

    Caution

    TBS is safe and non-toxic, requiring no special handling.

    References

    1. “Western Blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.
    2. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
    3. GE
    4. Termofisher
    5. Sigma
    6. Abcam
    7. Takara
    8. CSHL

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