Streptavidin-Xtra™ IF555
| Overview |  SDSProtocol |
See also: Bioconjugation, Biotin and Streptavidin
Molecular weight ~52000 | Correction Factor (260 nm) 0.23 | Correction Factor (280 nm) 0.14 | Extinction coefficient (cm -1 M -1) 1000001 | Excitation (nm) 557 | Emission (nm) 570 | Quantum yield 0.641 |
Streptavidin conjugates are widely used in combing with biotin conjugates for detecting a variety of biological targets such as proteins, nucleic acids and other molecules. They are used as an ideal choice for many biological detections such as immunofluorescence microscopy, flow cytometry, western blot and other biological applications since streptavidin has a strong affinity binding biotin which is not affected over a broad range of pH and temperature. AAT Bioquest® offers a variety of streptavidin conjugates labeled with the classic fluorescent dyes (for example: FITC, TRITC, Texas Red®, Cy3®, Cy5® and Cy7®) and also our superior water soluble, photostable iFluor® and mFluor™ dyes. However, the conventional biotin-avidin detection systems are still limited by the limited signal intensity of the existing fluorescent conjugates. The Streptavidin Xtra™ iFluor conjugates are a new family of super bright streptavidin conjugates with nearly identical excitation and emission properties to Alexa Fluor fluorophores with 3~5 folds signal improvement. It is a set of powerful tools to detect low abundance targets in cell imaging or flow cytometry. iFluor® 555 is one of the most common orange/red fluorescence colors for the Cy3 channel imaging.
Platform
Flow cytometer
| Excitation | 532 nm laser |
| Emission | 575/26 nm filter |
| Instrument specification(s) | PE channel |
Fluorescence microscope
| Excitation | Cy3/TRITC filter set |
| Emission | Cy3/TRITC filter set |
| Recommended plate | Black wall/clear bottom |
Example protocol
PREPARATION OF WORKING SOLUTION
Streptavidin-Xtra™ working solution
For IF, the suggested staining concentration is at 1-5 ug/ml. For FACS, the suggested concentration is at 0.1-0.5 ug / 100 uL / million cells in staining buffer. Note: PBS + 0.1% BSA can be used as a staining buffer. Note: For the best performance of each application, the optimal concentration of this reagent needs to be carefully determined. Note: The suggested working dilution is provided as a guide only. It is recommended that the users titrate the product in their tests using proper positive and negative controls.
SAMPLE EXPERIMENTAL PROTOCOL
- Block and treat the samples with antibodies of interest as per the manufacturer's recommendations.
- Add biotin conjugated secondary antibody working solution in the samples at appropriate concentration and duration. Note: Please verify the compatibility and type of your biotin conjugated antibody with the primary antibody used in the experiment. For example, If the primary antibody is Mouse antibody then Goat Anti-Mouse antibody bound with biotin can be used for the assay.
- Incubate the cells with Streptavidin-Xtra™ working solution at room temperature for 30 minutes to 1 hour. Note: Optimal time for incubation needs to be determined carefully.
- Remove the working solution and resuspend the cells in your choice of buffer.
- Take the image using the fluorescence microscope or record the intensity using flow cytometer.
Spectrum
Open in Advanced Spectrum Viewer
Spectral properties
| Correction Factor (260 nm) | 0.23 |
| Correction Factor (280 nm) | 0.14 |
| Extinction coefficient (cm -1 M -1) | 1000001 |
| Excitation (nm) | 557 |
| Emission (nm) | 570 |
| Quantum yield | 0.641 |
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
| Streptavidin-Xtra™ IF488 | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
| Streptavidin-Xtra™ IF594 | 568 | 587 | 1000001 | 0.571 | 0.34 | 0.15 |
| Streptavidin-Xtra™ IF647 | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
Images
Figure 1. Images of Hela cells stained with Streptavidin-Xtra™ iFluor® conjugates and Streptavidin Alexa Fluor ™ conjugate.
Hela cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.02% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour. Fixed Hela cells were then stained with 1 µg/mL alpha Tubulin Mouse Monoclonal Antibody for 1 hour at room temperature, followed by GxM IgG-biotin (Cat# 16729) stain and then visualized with Streptavidin-Xtra™ iFluor 555 and Streptavidin-Alexa Fluor™ 555 . Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).
Hela cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.02% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour. Fixed Hela cells were then stained with 1 µg/mL alpha Tubulin Mouse Monoclonal Antibody for 1 hour at room temperature, followed by GxM IgG-biotin (Cat# 16729) stain and then visualized with Streptavidin-Xtra™ iFluor 555 and Streptavidin-Alexa Fluor™ 555 . Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).
Citations
View all 35 citations: Citation Explorer
Highly sensitive electrochemical biosensor for streptavidin detection based on CdSe quantum dots
Authors: Wei, Y. P., Liu, X. P., Mao, C. J., Niu, H. L., Song, J. M., Jin, B. K.
Journal: Biosens Bioelectron (2018): 99-103
Authors: Wei, Y. P., Liu, X. P., Mao, C. J., Niu, H. L., Song, J. M., Jin, B. K.
Journal: Biosens Bioelectron (2018): 99-103
Efficient streptavidin-functionalized nitrogen-doped graphene for the development of highly sensitive electrochemical immunosensor
Authors: Yang, Z., Lan, Q., Li, J., Wu, J., Tang, Y., Hu, X.
Journal: Biosens Bioelectron (2017): 312-318
Authors: Yang, Z., Lan, Q., Li, J., Wu, J., Tang, Y., Hu, X.
Journal: Biosens Bioelectron (2017): 312-318
High-sensitive surface plasmon resonance microRNA biosensor based on streptavidin functionalized gold nanorods-assisted signal amplification
Authors: Hao, K., He, Y., Lu, H., Pu, S., Zhang, Y., Dong, H., Zhang, X.
Journal: Anal Chim Acta (2017): 114-120
Authors: Hao, K., He, Y., Lu, H., Pu, S., Zhang, Y., Dong, H., Zhang, X.
Journal: Anal Chim Acta (2017): 114-120
Correction to Peptide Tag-Induced Horseradish Peroxidase-Mediated Preparation of a Streptavidin-Immobilized Redox-Sensitive Hydrogel
Authors: Mishina, M., Minamihata, K., Moriyama, K., Nagamune, T.
Journal: Biomacromolecules (2017): 311
Authors: Mishina, M., Minamihata, K., Moriyama, K., Nagamune, T.
Journal: Biomacromolecules (2017): 311
DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA
Authors: Guo, Q., Han, J. J., Shan, S., Liu, D. F., Wu, S. S., Xiong, Y. H., Lai, W. H.
Journal: Biosens Bioelectron (2016): 990-995
Authors: Guo, Q., Han, J. J., Shan, S., Liu, D. F., Wu, S. S., Xiong, Y. H., Lai, W. H.
Journal: Biosens Bioelectron (2016): 990-995
Target-protecting dumbbell molecular probe against exonucleases digestion for sensitive detection of ATP and streptavidin
Authors: Chen, J., Liu, Y., Ji, X., He, Z.
Journal: Biosens Bioelectron (2016): 221-8
Authors: Chen, J., Liu, Y., Ji, X., He, Z.
Journal: Biosens Bioelectron (2016): 221-8
Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay
Authors: Lakshmipriya, T., Gopinath, S. C., Tang, T. H.
Journal: PLoS One (2016): e0151153
Authors: Lakshmipriya, T., Gopinath, S. C., Tang, T. H.
Journal: PLoS One (2016): e0151153
Peptide Tag-Induced Horseradish Peroxidase-Mediated Preparation of a Streptavidin-Immobilized Redox-Sensitive Hydrogel
Authors: Mishina, M., Minamihata, K., Moriyama, K., Nagamune, T.
Journal: Biomacromolecules (2016): 1978-84
Authors: Mishina, M., Minamihata, K., Moriyama, K., Nagamune, T.
Journal: Biomacromolecules (2016): 1978-84
Sensitive detection of platelet-specific antibodies with a modified MAIPA using biotinylated antibodies and streptavidin-coated beads
Authors: Mortberg, A., Meinke, S., Berg, P., Killie, M. K., Kjeldsen-Kragh, J., Jaras, K., Refsum, E., Hoglund, P., Wikman, A.
Journal: J Immunol Methods (2016): 9-15
Authors: Mortberg, A., Meinke, S., Berg, P., Killie, M. K., Kjeldsen-Kragh, J., Jaras, K., Refsum, E., Hoglund, P., Wikman, A.
Journal: J Immunol Methods (2016): 9-15
Facile fabrication of an electrochemical aptasensor based on magnetic electrode by using streptavidin modified magnetic beads for sensitive and specific detection of Hg(2.)
Authors: Wu, D., Wang, Y., Zhang, Y., Ma, H., Pang, X., Hu, L., Du, B., Wei, Q.
Journal: Biosens Bioelectron (2016): 9-13
Authors: Wu, D., Wang, Y., Zhang, Y., Ma, H., Pang, X., Hu, L., Du, B., Wei, Q.
Journal: Biosens Bioelectron (2016): 9-13