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Lidstrom:Buffers: Difference between revisions
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→Notes specific to buffers
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== Notes specific to buffers == | == Notes specific to buffers == | ||
=== phosphate buffer === | === phosphate buffer === | ||
* Phosphates, for example, form insoluble salts with bivalent metals and precipitate. Phosphate buffered salt solution (PBS) is never autoclaved with Ca2+ or Mg2+ for this reason. Good buffers, such as PIPES, TES, HEPES and CAPS have very low metal-binding constants and are therefore particularly suited to investigate metal-dependent enzymes (Good & Izawa 1972, Blanchard 1984). [http://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf Applichem] | *pH rane: 5.8-8 | ||
*Phosphates and pyrophosphate are both substrates and inhibitors of different enzyme reactions (inhibition of carboxypeptidase, urease, various kinases, various dehydrogenases). [http://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf Applichem] | * Phosphate buffering is based on the fact that phosphate can be in four states based on the pH. | ||
* Phosphates inhibit many enzymatic reactions and procedures that are the foundation of molecular cloning, including cleavage of DNA by many restriction enzymes, ligation of DNA, and bacterial transformation. ([http://www.unl.edu/cahoonlab/phosphate%20buffer.pdf source]) | ** [[image:2014_02_09 phosphate states]] | ||
* Because phosphates precipitate in ethanol, it is not possible to precipitate DNA and RNA from buffers that contain significant quantities of phosphate ions. ([http://www.unl.edu/cahoonlab/phosphate%20buffer.pdf source]) | ** It will shift between the different levels of protonation based on how many protons are available in solution. Thus it can be a source/sink for protons. Like all (good) buffers, if more H+ ions show up in solution, it can help absorb these and maintain the initial pH. Converseley, it can release H+ ions when they disappear from solution. | ||
* | *Aqueous phosphate exists in four forms. In strongly basic conditions, the phosphate ion (PO<sub>4</sub><sup>3−</sup>) predominates, whereas in weakly basic conditions, the hydrogen phosphate ion (HPO<sub>4</sub><sup>2−</sup>) is prevalent. In weakly acid conditions, the dihydrogen phosphate ion (H2PO<sub>4</sub><sup>−</sup>) is most common. In strongly acidic conditions, trihydrogen phosphate (H<sup>3</sup>PO<sub>4</sub>) is the main form. | ||
==== advantages ==== | |||
* Most physiological of common buffers. Mimics certain components of extracellular fluids. | |||
* Non-toxic to cells. | |||
* pH changes little with temperature. | |||
* Stable for several weeks at 4 C. | |||
==== disadvantages ==== | |||
* bind ions, and can cause precipitation: | |||
** Phosphates sequester divalent cations such as Ca2+ and Mg2+. ([http://www.unl.edu/cahoonlab/phosphate%20buffer.pdf source]) | |||
** Phosphates, for example, form insoluble salts with bivalent metals and precipitate. Phosphate buffered salt solution (PBS) is never autoclaved with Ca2+ or Mg2+ for this reason. Good buffers, such as PIPES, TES, HEPES and CAPS have very low metal-binding constants and are therefore particularly suited to investigate metal-dependent enzymes (Good & Izawa 1972, Blanchard 1984). [http://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf Applichem] | |||
* inhibit some enzymes | |||
**Phosphates and pyrophosphate are both substrates and inhibitors of different enzyme reactions (inhibition of carboxypeptidase, urease, various kinases, various dehydrogenases). [http://www.applichem.com/fileadmin/Broschueren/BioBuffer.pdf Applichem] | |||
** Phosphates inhibit many enzymatic reactions and procedures that are the foundation of molecular cloning, including cleavage of DNA by many restriction enzymes, ligation of DNA, and bacterial transformation. ([http://www.unl.edu/cahoonlab/phosphate%20buffer.pdf source]) | |||
* can precipitate | |||
** Because phosphates precipitate in ethanol, it is not possible to precipitate DNA and RNA from buffers that contain significant quantities of phosphate ions. ([http://www.unl.edu/cahoonlab/phosphate%20buffer.pdf source]) | |||
=== Sodium versus Potassium phosphate buffers === | |||
* The principle of phosphate buffering is independent of the source of phosphates. All that matters for buffering is the ratio of the H2PO4 and HPO4 ions in solution. | |||
* You can buy the | |||
You can get this by starting with the correct proportion, or by forcing the equilibrium to shift by adding acid or base. How you get this can vary. You can add various amounts of the two ions; both are available as sodium and potassium salts | |||
=== Tris buffer === | === Tris buffer === | ||
