buffers lecture
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Buffers LectureTRANSCRIPT
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The pKa of the buffer should be within 0.5 unit of the desired pH (± 1 unit if you want to push it)
Potential interactions with a column matrix
Avoid UV-absorbing buffers if you plan to use a UV detector
The ionic strength and salt composition must be chosen according to the stability of the protein and the detergent
Choice of Buffer
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Hydrogen Ion Buffers for Biological Research*Norman E. Good, G. Douglas Winget, Wilhelmina Winter,Thomas N. Connolly, Seikichi Izawa, and Raizada M. M. Singh Biochemistry, 1966, 5 (2), 467-477• DOI:
In other words, you want a “Good” buffer:
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Biochemistry, 1966, 5 (2), 467-477
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Biochemistry, 1966, 5 (2), 467-477
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Biochemistry, 1966, 5 (2), 467-477
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Biochemistry, 1966, 5 (2), 467-477
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Preparation of Buffers
How would one make 1 L of a 2.0 M stock solution ofTris·Cl at pH 8.0?
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How would one make 1 L of a 1.0 M stock solution ofK+·MES at pH 6.5?
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Many enzymes require a particular metal ion for optimalactivity, and many of these enzymes are inhibited by metal ions other than their physiological, activating ones.
What treatment should be done to a buffer solution toensure that the buffer is free of contaminating metal ions?
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Let us say that you want to do a pH profile of an enzyme(i.e., assay the log(activity) of the enzyme as a function ofpH. What precautions should you take before you engagein such a study?
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Let us say that the product of the enzyme-catalyzed reaction that you are studying as a function of pH absorbsat a particular wavelength, say 240 nm, but the substratedoes not absorb appreciably at this wavelength. Are there any concerns about the λmax of the chromophoreproduct as the pH changes?
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Let us say that the molar extinction coefficient of the chromophore product does change with pH. It would be nice if one could derive a relationship between the molar extinction coefficient and pH (i.e., how the extinctioncoefficient changes as a function of pH). Hint: we did a similar derivation when we developed the theory behind the “pKa of Indicator” lab. OK, so give it a try...