New PDF release: A Laboratory Guide to Biotin-Labeling in Biomolecule

By M. Dean Savage (auth.), Dr. Thomas Meier, Dr. Falk Fahrenholz (eds.)

ISBN-10: 3034873492

ISBN-13: 9783034873499

ISBN-10: 3034873514

ISBN-13: 9783034873512

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Additional info for A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis

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H I-SH + "";::r ft ft o II HNG~"H 5 {)1 pH l . 5 Reaction of biotin-HPDP with a sulfhydryl 17 M. 7 Reaction of biotin-BMCC with a sulfhydryl Considerations for the Use of Sulfhydryl-Reactive Biotinylation Reagents Most commonly, the source of the sulfhydryl group for biotinylation is the cysteine on the surface of a protein or within a peptide. If a sulfhydryl group is not available, it can be introduced through prior reaction of the macromolecule with the use of SATA (46), Traut's reagent (47), or SPDP (32).

3 ensures the modification of sulfhydryl groups and not amino groups. Histidyl side chains and amino groups react in the unprotonated form and may be observed to take part in reactions above pH 5 and pH 7, respectively, although this reaction is much slower than that for sulfhydryls (50). The mercaptoethylamine reduction protocol works well with a variety of polyclonal antibodies. It can easily be optimized by varying the time of reduction. Aliquots can be pulled and subjected to nonreducing electrophoresis and the frag- 26 Introduction to Avidin-Biotin Technology and Options for Biotinylation ments analyzed.

Fol low with a thorough homogenization using a Polytron homogen izer (Kinematica GmbH). 3. Centrifuge the homogenate at 10000 x g for 15 min to ob- ta in the supernatant. 4. Ultracentrifuge the supernatant at 100 000 x g for 1 hour. 5. 4). Ultracentrifuge the suspension aga in. 6. 15 M NaCI-TEDs buffer and once with TEDs buffer. 7. Suspend the membrane pellet in TEDs buffer at 7 to 10 mg protein/ml. Store it at -70 ·C. 50 Purification of the Receptor for Pituitary Adenylate Cyclase-Activating ..

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A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis by M. Dean Savage (auth.), Dr. Thomas Meier, Dr. Falk Fahrenholz (eds.)

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