Quanta BioDesign’s cleavable biotin reagent NHS-S-S-dPEG®4-biotin, product number 10194, is designed for reversible biotin labeling of biomolecules and surfaces. Combining the distinctive, beneficial characteristics of NHS-dPEG®4-biotin (PN10200) with an additional, cleavable disulfide spacer creates a novel amine-reactive biotinylation reagent. Any biomolecule or surface with one or more free amines can be labeled with this reagent. When needed, mild reductive conditions break the disulfide bond, releasing the label. See Figure 1, below.
Cleavable Biotin for Antibody-Antigen Purification
One type of application where NHS-S-S-dPEG®4-biotin is useful involves biotin-labeled antibodies. In this type of application, first, an antibody labeled with NHS-S-S-dPEG®4-biotin binds to its target antigen. Second, affinity chromatography using immobilized streptavidin purifies the antibody-antigen complex. Third, mild reduction using DTT or TCEP releases the antigen-antibody complex from streptavidin. Fourth and last, the complex elutes from the column.[1],[2],[3] Combined with phage display, this type of application has
- isolated antibody inhibitors of the binding sites of paxillin[4] and Cyclophilin A[5];
- generated synthetic antibodies to structural, membrane-bound proteins[6]; and,
- determined how cells sense voltage changes[7].
Also, a noncompetitive immunocomplex immunoassay for screening a broad spectrum of small molecular weight analytes (<2000 Daltons) used NHS-S-S-dPEG®4-biotin as a component of the screening system[8].
Enzyme Activity Analysis Using Cleavable Biotin
Another type of application uses GTP labeled with NHS-S-S-dPEG®4-biotin and an antibody fragment specific to guanosine triphosphate (GTP) to analyze GTPase activity using a technique known as quenching resonance energy transfer (QRET)[9]. GTPase enzymes such as RAS often are implicated in cancer. By using GTP-specific antibodies, this method effectively finds GTPase inhibitors. (Antibodies that cross-react with other nucleotides such as GDP or ATP are unsuitable.)
GTP biotinylated with NHS-S-S-dPEG®4-biotin is added to a GTPase signaling system followed by Eu3+-labeled streptavidin. Immobilized anti-GTP Fab binds the GTP-biotin-Eu3+-streptavidin complex. When illuminated at 340 nm, the complex fluoresces at 615 nm. Following the addition of all components of the GTPase system, GTP hydrolyzes to GDP, and the signal is lost. Potential GTPase inhibitors are identified by adding them to the system and seeing whether a possible GTPase inhibitor prevents hydrolysis and consequent loss of fluorescence.[10],[11],[12]
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References
[1] Hermanson, G. Chapter 9. Cross-Bridges and Cleavable Reagent Systems. In Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, pages 387-394. Most bioconjugation experts consider Greg Hermanson’s book to be one of the best references for this field. Please click here for a review of this book and to purchase it.
[2] Hermanson, G. Section 6. Biotinylation Techniques. In Chapter 11. (Strept)avidin-Biotin Systems. In Bioconjugate Techniques, 3rd edition, pages 479-490.
[3] Hermanson, G. Section 1.3 Biotinylation Reagents Containing Discrete PEG Linkers. In Chapter 18. PEGylation and Synthetic Polymer Modification. In Bioconjugate Techniques, 3rd edition, pages 806-821.
[4] Nocula-Lugowska, M.; Lugowski, M.; Salgia, R.; Kossiakoff, A. A. Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin. J. Mol. Biol. 2015, 427(15), 2532-2547. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521624/.
[5] Zheng, J. Multidisciplinary Analysis of Cyclophilin A Function in Human Breast Cancer. Annual summary 1 Mar 2008-28 Feb 2011. Technical report for Defense Technical Information Center, Award Number W81XWH-08-0169. Accession Number: ADA543741. March 1, 2011.
[6] Dominik, P. K.; Borowska, M. T.; Dalmas, O.; Kim, S. S.; Perozo, E.; Keenan, R. J.; Kossiakof, A. A. Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs. Structure 2016, 24(2), 300-309. https://www.cell.com/structure/fulltext/S0969-2126(15)00503-1.
[7] Li, Q.; Wanderling, S.; Paduch, M.; Medovoy, D.; Singharoy, A.; McGreevy, R.; Villalba-Galea, C.; Hulse, R. E.; Roux, B.; Schulten, K.; Kossiakoff, A.; Perozo, E. Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain. Nat. Struct. Mol. Biol. 2014, 21(3), 244-252. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116111/.
[8] Akter, S.; Vehniainen, M.; Spoof, L.; Nybom, S.; Meriluoto, J.; Lamminmaki, U. Broad-spectrum noncompetitive immunocomplex immunoassay for cyanobacterial peptide hepatotoxins (microcystins and nodularins). Anal. Chem. 2016, 88, 10080-10087. https://doi.org/10.1021/acs.analchem.6b02470.
[9] Kopra, K.; Harma, H. Quenching resonance energy transfer (QRET): A single-label technique for inhibitor screening and interaction studies. New Biotechnology 2015, 32(6), 575-580. https://www.sciencedirect.com/science/article/pii/S1871678415000242.
[10] Kopra, K. From Unspecific Quenching to Specific Signaling: Functional GTPase Assays Utilizing Quenching Resonance Energy Transfer (QRET) Technology. Ph.D. Dissertation. University of Turku. 2015, April 1, 2015. ISBN 978-951-29-6080-4.
[11] Kopra, K.; Ligabue, A.; Wang, Q.; Syrjanpaa, M.; Blazevits, O.; Veltel, S. van Adrichem, A. J.; Hanninen, P.; Abankwa, D.; Harma, H. A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction. Anal. Bioanal. Chem. 2014, 406, 4147-4156. https://www.ncbi.nlm.nih.gov/pubmed/24760397.
[12] Kopra, K.; Rozwandowicz-Jansen, A.; Syrjanpaa, M.; Blazevits, O.; Ligabue, A.; Veltel, S.; Lamminimaki, U.; Abankwa, D.; Harma, H. GTP-specific Fab fragment-based GTPase activity assay. Anal. Chem. 2015, 87, 3527-3534. https://www.ncbi.nlm.nih.gov/pubmed/25707436.