Streptavidin Columns

The challenge of modern Target Discovery requires the efficient automation of complex assays. The immunoprecipitation (IP) assay has proven quite useful in research discovery to identify components of molecular complexes, discovering protein-protein and protein-nucleic acid interactions, and quantifying an immunogenic response.

Protein A PhyTip columns have been successfully used for this purpose, but IP can only be successful if an antibody is available to the peptide or protein of interest. The availability of biotin labeling reagents allows researchers to generate customized affinity resins by conjugating proteins of interest to streptavidin resins. These custom resins are then ready to pull down interactive proteins and complexes from biological samples. To efficiently automate these assays, significant sample preparation steps must be addressed. Namely, protein needs to be purified, excess biotin labeling reagent must be removed, and biotinylated protein needs to be immobilized.

PhyNexus Affinity Purification, Gel Filtration columns containing resin of 5K molecular weight cutoff, and Streptavidin columns in combination are the ideal tools for small-volume, automated, high throughput purification, separation, and immobilization of biotinylated proteins for complex assays.

Streptavidin columns with our proprietary Dual Flow Chromatography provide complete antibody binding and high final concentrations. Streptavidin tips are compatible with most 8, 12, and 96-channel automation liquid handling robots including Agilent TechnologiesBeckman CoulterDynamic DevicesPerkin ElmerHamilton, and Tecan.

Description

PhyTip columns are unique capture, purification and enrichment™ tools from PhyNexus designed for lab-scale protein sample preparation. PhyTip columns are available for a variety of liquid handling platforms and contain specific affinity resins for application specific requirements.

PhyNexus recommends the use of biotin-labeled bovine albumin (Sigma, A8549) as a standard to verify the use of PhyTip columns with all applications.

Samples for purification and enrichment must be clear and free from particulate matter. It is highly recommended to centrifuge samples and use the clear supernatant only, prior to use with PhyTip columns.

Application

PhyTip columns with Streptavidin PhyTip columns with Streptavidin have been optimized for use with general molecular biology laboratory reagents and specific PhyNexus instrument flow rates/volumes as shown below. This information was collected using the PhyNexus Purification Systems.

PhyNexus recommends the following buffers (not supplied):

Capture Buffer – Phosphate Buffer solution pH 7.4, for those situations where additional buffer needs to be added to supplement the volume of the sample and to ensure correct pH for capture

Wash Buffer I – Phosphate Buffer solution pH 7.4

Wash Buffer II – Phosphate Buffer solution pH 7.4

Enrichment Buffer – If performing a pull-down assay, for the final elution step may be compatible with an acidic buffer such as Phosphate Buffer solution pH 2.5

Neutralization Buffer. – Tris Buffer solution pH 9.0

Data

PhyNexus recommends the use of the PhyTip Starter Kit containing IgG as a standard to verify the use of PhyTip columns with all applications.

Samples for purification and enrichment must be clear and free from particulate matter. It is highly recommended to centrifuge samples and use the clear supernatant only, prior to use with PhyTip columns.

Shipping and Storage

Each pack of PhyTip columns has been manufactured and QC’d to the highest standards and shipped in retainer boxes that maintain the integrity of the specific affinity resin within each PhyTip column. This product is shipped at ambient temperatures, but on receipt should be stored in a standard laboratory refrigerator between 4 and 8◦C.

  • Do NOT freeze or store frozen.
  • When not in use, keep the lid of the box closed and sealed, store in the refrigerator.
  • Do not allow affinity resin to dry out by extended storage in a dry environment.

PhyTip columns with Streptavidin are stored in Glycerol when shipped from PhyNexus.

Documents
Compatibility

Biotin-tagged PhyTip Columns are available for these robotic systems.

Citations

Publications


Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase
Concha N, Huang J, Bai X, Benowitz A, Brady P, Grady L, Kryn L, Holmes D, Ingraham K, Jin Q, Kaushansky L, McCloskey L, Messer J, O’Keefe H, Patel A, Satz A, Sinnamon R, Schneck J, Skinner S, Summerfield J, Taylor A, Taylor J, Evindar G, Stavenger R
Publication: Journal of Medicinal Chemistry, 2016
doi: 10.1021/acs.jmedchem.6b00746
pubs.acs.org/doi/abs/10.1021/acs.jmedchem.6b00746

Analysis of the Productivity of DNA Encoded Libraries
Eidam O, Satz A
Publication: Med. Chem. Commun., 2016
doi: 10.1039/C6MD00221H
pubs.rsc.org/en/Content/ArticleLanding/2016/MD/C6MD00221H

Encoded library technology as a source of hits for the discovery and lead optimization of a potent and selective class of bactericidal direct inhibitors of mycobacterium tuberculosis inha
Encinas L, O’Keefe H, Neu M, Remuiñán M, Patel A, Guardia A, Davie C, Pérez-Macías N, Yang H, Convery M, Messer J, Pérez-Herrán E, Centrella P, Álvarez-Gómez D, Clark M, Huss S, O’Donovan G, Ortega-Muro F, McDowell W, Castañeda P, Arico-Muendel C, Pajk S, Rullás J, Angulo-Barturen I, Álvarez-Ruíz E, Mendoza-Losana A, Ballell Pages L, Castro-Pichel J, Evindar G, GlaxoSmithKline, Consejo Superior de Investigaciones Cientificas, X-Chem
Publication: Journal of Medicinal Chemistry, 2014 vol: 57 (4) pp: 1276-1288
doi: 10.1021/jm401326j
https://www.ncbi.nlm.nih.gov/pubmed/24450589

Posters


Integration of Automated Bead-and Cartridge-Based Immunocapture Workflows on a Generic Tecan Platform for LC-MS Bioanalysis of Protein Therapeutics and Biomarkers
Naiyu Zheng, Huidong Gu, Kristin Taylor, Yan J. Zhang, Jianing Zeng (Analytical and Bioanalytical Operations, Bristol-Myers Squibb Company, Princeton, NJ)

Screening for Chromatographic Success and Failure on Small Scale
W. Gillette (NCI/SAIC-Frederick)

Space is the Place: Using DOE and Microscale Techniques to Define Process Boundaries
M. Wenger (Merck & Co.)

Development of Small-Scale, High Throughput, Chromatographic Protein Purification
K. Valente1, M. Wenger2, J. Shanter1, S. Sagar1, J. Konz1 (1Merck Biopurification Development, 2Merck Bioprocess and Bioanalytical Research)

A flexible platform for automated high-performance protein purification using micro scale separation technology
J. Lambert, M. Anderson, D. Gjerde, L. Nguyen (PhyNexus)

Innovative Solutions for Automated Protein and Nucleic Acid Purification
Lee Hoang, PhyNexus, Inc., PhyNexus Webinar

Automated, High Throughput Protein Purification and Sample Prep Using PhyTip Columns for Therapeutic Leads Screening and Process Development
Lee Hoang, PhD., PhyNexus, Inc., The 5th Annual Proteins Congress 2012, London

Optimization of Protein Purification Using Small-Scale Separation Columns
Chris Suh, PhD., PhyNexus, Inc., Protein Therapeutics Discovery and Development

PhyNexus Users Group Symposium 2014
Douglas Gjerde, PhyNexus
Lee Hoang, PhyNexus
Tyson Bowen PacBio

Select your resin, robotic system, tip and resin volume and see the results in the table below.

Streptavidin PhyTip Columns