Download PDF by COLIGAN: Current Protocols in Protein Science


ISBN-10: 0471111848

ISBN-13: 9780471111849

ISBN-10: 0471140988

ISBN-13: 9780471140986

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After use, the GTP-Sepharose gel can be recycled as described in Support Protocol 4. ASSESSMENT OF INCORPORATION OF GTP INTO THE HYDRAZIDE GEL The following modification to Support Protocol 1 may be applied if there are any problems using the GTP-Sepharose medium prepared in Support Protocol 1 in the affinity purification steps (see Basic Protocol 1, steps 13 to 16). It is generally not necessary to resort to this procedure. SUPPORT PROTOCOL 2 CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings.

Take an aliquot of the enzyme-containing solution from the DEAE-cellulose purification step (see Alternate Protocol and Basic Protocol 1) and assay it for GDH activity (see Basic Protocol 3) and protein concentration (see Basic Protocol 2) to determine its specific activity. 6 An example of a pilot-scale affinity precipitation of ox liver glutamate dehydrogenase purified according to steps 1 to 3 of the Alternate Protocol. The experimental details are described in Support Protocol 9. The precise behavior will vary between preparations.

Store at 4°C with an overlay of butanol, to retard bacterial growth. Remove the butanol layer by aspiration and equilibrate with affinity chromatography buffer (see recipe) immediately before use. GTP Sepharose (prepared as in Support Protocol 1, steps 1 to 7) Follow the same procedure as described for the hydrazide-Sepharose, above. Over a period of several months, the properties of the GTP-Sepharose change and the enzyme activity is eluted earlier in the gradient, probably as a result of instability of the bound GTP.

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Current Protocols in Protein Science by COLIGAN

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