Sodium acetate buffer

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    Introduction

    Various authors (Gomori, 1955; Pearse 1980) are cited as a source for buffer composition, although it should be said that probably the first author specifying this buffer was Walpole (1914). However, the composition of the cited buffers is really similar. Thanks to the pH variability and composition of available compounds, it is sometimes the first choice for many reactions.

    The sodium acetate buffer could be replaced with various other buffers (depending on application) as mentioned below.

    • For use in animal physiology / medical use, replacement by sodium bicarbonate buffer is sometimes possible (Neavyn et al., 2013).
    • In enzymology, it could be replaced by citrate, which has a similar range of pH but lowers buffering capacity.
    • In nucleic acid precipitation protocols by sodium chloride (preferred in detergents),
    • Ammonium acetate (in high dNTPs, without kinases),
    • Potassium acetate (for cell-free translation, avoid SDS),
    • Lithium chloride (useful in RNA, avoid in translation) (Genelink).
    • When using acetate for protein crystallization, it has as wide application as ammonium sulfate but it was found to be less effective than sodium malonate (Mcpherson, 2001).

    Applications

    • This buffer is used in several enzymes using protocols enabling them to work in mildly acidic pH.
    • Other important protocols utilizing sodium acetate buffer are purification and precipitation of nucleic acids, protein crystallization, and staining of gels after protein gel electrophoresis.

    pH

    For enzymology and chromatography: In the range from 3.6 to 5.6.

    For precipitation of nucleic acids: 3 M stock with pH from 5.2 to 5.5. pH is adjusted by glacial acetic acid / NaOH.

    For protein crystallization: Various pH used based on the need of protein. Some reported pHs are 4.0-4.9 for x-ray crystallography (Dessau and Modis, 2011) and 7.2 (Mcpherson, 2001). pH is adjusted by glacial acetic acid / NaOH.

    For gel silver staining: 0.4 M solution with pH 6.0 (Heukeshoven and Dernick, 1988).

    Composition

    For enzymology or chromatography

    ReagentMolecular weight1X molarityAdd for 500ml of 1XAdd for 1L of 1X
    Sodium acetate82.03 (anhydrous)100 mM4.1 g8.2 g
    Sodium acetate136.08 (trihydrate)100 mM6.8 g13.6 g
    Acetic acid (glacial)60.05100 mM3.0 g (2.9 ml)6.0 g (5.8 ml)

    For precipitation

    ReagentMolecular weight1X molarity10X molarityAdd for 100 ml of 10XAdd for 1L of 10X
    Sodium acetate82.03 (anhydrous)300 mM3 M26.6 g246 g
    Sodium acetate136.08 (trihydrate)300 mM3 M40.8 g408 g

    For staining

    ReagentMolecular weight1X molarity10X molarityAdd for 100 ml of 10XAdd for 1L of 10X
    Sodium acetate82.03 (anhydrous)400 mM4 M32.8 g328 g
    Sodium acetate136.08 (trihydrate)400 mM4 M54.4 g544 g

    Non-factorable compounds

    ReagentMolecular weight1X molarityAdd for 100 ml of 1XAdd for 1 l of 1X
    Acetic acid (glacial)60.0575 mM0.435 g (0.45 ml)4.35 g (4.5 ml)
    Ethanol46.07-0.330 ml300 ml
    Glutaraldehyde (25%)100.1250 mM (0.5%)2 ml20 ml
    Sodium thiosulfate158.116.3 mM (0.1%)0.1 g1 g

    Reagents

    Sodium acetate (anhydrous or trihydrate).

    Acetic acid (glacial).

    Ethanol, glutaraldehyde, sodium thiosulfate (if needed for staining).

    Acetic acid and NaOH could be used for pH adjustment.

    Instruments and other requirements

    Glass beaker

    Weighing balance

    Magnetic stirrer and pellet

    pH meter

    Measuring cylinders

    Pipette

    Procedure

    1. For enzymology and chromatography, 1 L of the final solution:

    Firstly, prepare the stock solution of sodium acetate:

    Add about ¾ of the final volume of distilled water to a glass beaker.

    Prepare the exact amounts of sodium acetate (either anhydrous or trihydrate) using balance and put it into the water to dissolve. A magnetic stirrer can be used to speed up the process.

    After the acetate is dissolved, add distilled water to the final volume.

     

    Secondly, prepare the stock of acetic acid with similar steps:

    Add about ¾ of the final volume of distilled water to the glass beaker.

    Prepare the exact amounts of acetic acid (glacial) volumetrically (or gravimetrically) and put it into the water to mix well.

    Add distilled water to the final volume.

    Autoclave the solutions.

    In the next step, mix specific amounts of sodium acetate solution and acetic acid solution according to the table below to obtain the buffer of the required pH.

    2. For precipitation and staining

    Firstly, prepare stock (10x) of sodium acetate:

    Add about ¾ of the final volume of distilled water to a glass beaker.

    Prepare the exact amounts of sodium acetate (anhydrous or trihydrate) using balance and put it into the water to dissolve. A magnetic stirrer can be used to speed up the process.

    After the sodium acetate is dissolved, adjust the pH with acetic acid (glacial) or NaOH.

    Add distilled water to the final volume.

    (It is sometimes recommended to let the solution stay for a day and recheck the pH / adjust again).

    Autoclave this solution/filter it.

    If you are willing to use this buffer for precipitation, you just end with preparation here and you can dilute your stock solution 10x when needed, but for staining the solution contains additional compounds, which will be added in the next step.

    In the last step, prepare the solution for staining use (1l):

    Mix ethanol (300 ml) with sodium acetate stock (100 ml) solution and add distilled water to the final volume. (Recheck the pH; It should be 6,0; adjust with glacial acid)

    Add the exact volume of glutaraldehyde (Toxic!) and the exact amount of sodium thiosulfate. Let it dissolve and mix well.

    Storage

    It can be stored at room temperature or at 4° C (better for less concentrated solutions to avoid precipitation). If you added thiosulfate, store it only for a few days.

    Caution

    Glutaraldehyde is a toxic compound, similar to other aldehydes. Wear gloves and glasses. Omit skin or mucosa contact.

    Glacial acid is a highly concentrated acetic acid; thus, you should treat it as carefully as with strong acids. Omit any skin or eye contact.

    Reference

    1. Acetate Buffer (pH 3.6 to 5.6) Preparation and Recipe | AAT Bioquest.
    2. Dessau, M. A., & Modis, Y. (2011). Protein crystallization for X-ray crystallography. JoVE (Journal of Visualized Experiments), (47), e2285.
    3. Gomori, G. (1955). [16] Preparation of buffers for use in enzyme studies.
    4. Heukeshoven, J., & Dernick, R. (1988). Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis9(1), 28-32.
    5. DNA & RNA Precipitation Solutions.
    6. Mcpherson, A. (2001). A comparison of salts for the crystallization of macromolecules. Protein Science10(2), 418-422.
    7. Neavyn, M. J., Boyer, E. W., Bird, S. B., & Babu, K. M. (2013). Sodium acetate as a replacement for sodium bicarbonate in medical toxicology: a review. Journal of Medical Toxicology9(3), 250-254.
    8. Pearse, A. G. E. (1980). Histochemistry, Vol. 1–2: Theoretical and applied. Churchill Livingstone, Edimburgh, London.
    9. Snyder, L. R., Kirkland, J. J., & Glajch, J. L. (2012). Practical HPLC method development. John Wiley & Sons.
    10. Sodium acetate buffer solution for molecular biology (Sigma-Aldrich).
    11. Walpole, G. S. (1914). CCXXXIII.—Hydrogen potentials of mixtures of acetic acid and sodium acetate. Journal of the Chemical Society, Transactions105, 2501-2521.