By Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby, Meir Wilchek
The severely acclaimed laboratory usual, Methods in Enzymology, is without doubt one of the such a lot hugely revered guides within the box of biochemistry. because 1955, each one quantity has been eagerly awaited, often consulted, and praised by means of researchers and reviewers alike. The sequence includes a lot fabric nonetheless appropriate this day - actually a necessary booklet for researchers in all fields of existence sciences
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Extra resources for Affinity Techniques - Enzyme Purification: Part B
Shaking of the funnel is continued vigorously for 3 minutes then less so for 7 more minutes. The lower layer is separated 23Glutaric anhydride (not to be confused with glutaraldehyde) can be purchased from Aldrich Chemical Co. (Milwaukee, Wisconsin) or Eastman Organic Chemicals (from their various distributors). Darkly colored material should be purified as follows: Dissolve 25 g of the crude anhydride in 125 ml of benzene. 5 g of activated charcoal and stir for 1 hour at room temperature. Filter and reduce the volume of filtrate to about 60 ml by boiling (in a hood).
For example, at a reaction time of 5 hours with 1 ml of reagent, the concentration of the oxirane groups in the activated ether will be about 200 t moles per gram of dry gel. The coupling should be performed in an alkaline solution at a pH be- 26 COUPLINGREACTIONS AND GENERAL METHODOLOGY  tween 11 and 12, if possible. At a lower pH the reaction proceeds more slowly and in an extremely alkaline medium, > p H ] 3, cross-linking is likely to lower the yield. 2 M sodium carbonate at pH 11, containing 300 mg of glycylleucine.
However, relatively few opportunities exist for subsequent reactions in aqueous media. 16 So far, water-soluble carbodiimides have been the reagents of choice in effecting subsequent reactions with the carboxyl function. The acyl hydrazide derivative ( I I ) is also easy to prepare but provides m a n y opportunities for the next stage of derivatization. It can be i, The degree of swelling is reproducible only if the polymer bears very few charged groups, or if it swells in solutions possessing enough electrolyte to effectively shield bound charges.
Affinity Techniques - Enzyme Purification: Part B by Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby, Meir Wilchek