Download Affinity Techniques - Enzyme Purification: Part B by Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby, PDF

By Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby, Meir Wilchek

The significantly acclaimed laboratory commonplace, Methods in Enzymology, is without doubt one of the such a lot hugely revered courses within the box of biochemistry. when you consider that 1955, each one quantity has been eagerly awaited, often consulted, and praised by way of researchers and reviewers alike. The sequence comprises a lot fabric nonetheless proper this present day - actually a necessary book for researchers in all fields of existence sciences

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Extra info for Affinity Techniques - Enzyme Purification: Part B

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Among the excellent properties of agarose should be mentioned the resistance to bacteria and enzymes likely to be present in the laboratory. No "agarase" has yet been found to cause an observable attack on cross-linked agarose. Acid sensitivity as manifested at low pH ( < 2 . 5 ) is a weak point. Rigidity is higher than for many other carrier materials, but an even greater rigidity would be desirable. Much harder gels can be obtained by cross-linking with divinylsulfone. Agarose cross-linked by this reagent retains high permeability, but unfortunately--owing to alkaline instability of the ether linkages--the working range for the divinylsulfone cross-linked agarose is limited to a pH below 9.

If the test is positive, showing unreacted hydrazide, then more glutaric anhydride is added and the 15-minute, p H 4 reaction is repeated. The gel is finally washed alternately by funnel and batch techniques for several cycles. Preparation of 6-Bromoacetamidocaproic Acid (BACA, VII). B A C A is a useful intermediate for introducing spaced-out b r o m o a c e t y l and a-amino groups. 10 m o l e ) of bromoacetic acid in 330 ml of ethyl acetate. 10 mole) of b r o m o a c e t y l bromide is added, z6 The funnel is shaken for about 1 minute (releasing excess v a p o r pressure) and allowed to stand for 1 0 - 1 5 minutes.

The dry beads are added gradually with stirring to an aqueous solution of hydrazine (1 M to 6 M ) . lr The suspension is stirred slowly or swirled occasionally while being held at a constant temperature. The reaction is stopped after the desired time by funnel washing of the gel. 2 M NaC1 are used if flow rates permit. Washing is completed by a series of batch treatfnents until supernatants (free of beads) become negative or show only pale violet color in the TNBS test. The gel should give a positive color reaction.

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