Streptavidin Sigma CAS 9013-20-1

1. Get to know streptavidin
Streptavidin (SA), a secreted protein derived from Streptomyces avidinii, has a molecular weight of approximately 65-66 kDa and an isoelectric point of around 6.0. It is a tetramer protein composed of four homologous subunits, each with an independent biotin binding site - meaning that one molecule of streptavidin can simultaneously bind to four molecules of biotin. The dissociation constant (Kd) between the two is as low as about 10 ⁻¹⁵ M, which is one of the strongest known non covalent biological interactions and can be regarded as the most precise "molecular locking mechanism" in nature.

Compared with avidin derived from egg white, it has unparalleled advantages:

It does not contain glycosylation modifications, is overall electrically neutral, therefore has extremely low non-specific binding under physiological conditions, clean experimental background, and excellent signal-to-noise ratio. In addition, its excellent resistance to denaturation and protease degradation, as well as its ability to withstand extreme pH, high temperatures (70-80 ° C), organic solvents, and conventional denaturing agents (such as SDS and urea), have replaced avidin and become the undisputed "gold standard" in biotin binding systems.

It is precisely with these outstanding characteristics that it shines in fields such as life sciences, in vitro diagnostics, drug screening, and biosensing.

2. Immune testing
The application in the field of immune detection is its most classic and widely used application. Since its development in the late 1970s, the biotin streptavidin system (BAS) has become the cornerstone of immune labeling and tracing analysis due to its ultra-high affinity and cascade amplification effect. According to statistics, about 85% of common chemiluminescence analyzers use BAS based detection methods, indicating their dominant position.

2.1 Enzyme linked immunosorbent assay (ELISA)

In ELISA, horseradish peroxidase (HRP) or alkaline phosphatase (AP) can be coupled and the enzyme can be precisely guided to the target through biotinylated antibodies. HRP catalyzes TMB, DAB, or ECL luminescent substrates to produce potent chemical or optical signals, while AP catalyzes BCIP/NBT, PNPP and other substrates to produce stable color or chemiluminescence signals. A biotinylated target molecule can bind to multiple labeled streptavidin, achieving multi-level signal amplification and making detection of low abundance targets possible. This technology is widely used in sandwich and competitive ELISA, covering scenarios such as protein quantification, antibody detection, pathogen screening, etc.

2.2 Protein immunoblotting (Western Blot)
In Western Blot, binding with biotinylated secondary antibodies can significantly enhance the signal intensity of target proteins on transfer films. Compared to traditional secondary antibody systems, BAS systems can achieve stronger signal amplification due to their tetravalent binding properties. Additionally, due to the low non-specific binding of streptavidin, background interference is significantly reduced, making them particularly suitable for quantitative analysis of low abundance proteins.

2.3 Immunohistochemistry (IHC) and Immunocytochemistry (ICC)
HRP labeled or AP labeled streptavidin is the preferred signal amplification tool for IHC and ICC. The HRP system is suitable for experiments that require extremely high detection sensitivity, while the AP system is particularly suitable for systems containing HRP inhibitors (such as sodium azide) or experimental scenarios that require long-term color development and signal stability. By binding biotinylated probes or antibodies to streptavidin conjugates, target molecules can be accurately located in tissue sections or cell samples.

2.4 Immunofluorescence (IF) and in situ hybridization (ISH)

Fluorescein labeled streptavidin (such as FITC Streptavidin, PE Streptavidin, APC Streptavidin) is a universal visualization tool for immunofluorescence staining and in situ hybridization. The FITC marker has an excitation/emission wavelength of approximately 494/519 nm and exhibits bright green fluorescence; The excitation/emission wavelength of PE marker is about 565/578 nm, which is the "gold standard" for multi-color flow cytometry analysis; The APC marker covers the far red spectrum, facilitating multi-color imaging. These fluorescent markers can accurately bind to biotinylated antibodies or nucleic acid probes to achieve localization analysis and quantitative detection of target molecules.

2.5 Flow cytometry (FACS)

In flow cytometry, fluorescently labeled streptavidin (especially PE labeled) is used to detect cell surface antigens labeled with biotinylated antibodies. Its excellent brightness provides an extremely high signal-to-noise ratio, which can effectively detect cell populations with low surface antigen expression or weak signals. It is an indispensable reagent in multi-color flow cytometry analysis.

2.6 Enzyme linked Immunospot Assay (ELISPOT)
AP labeled streptavidin exhibits excellent performance in ELISPOT and is suitable for solid-phase analysis and tissue/cell staining systems. It can stably catalyze substrate production of countable spot signals for single-cell level cytokine secretion detection.

3. Purification of proteins and nucleic acids
3.1 Affinity chromatography and protein purification
Streptomycin is the core component of affinity chromatography packing. After biotin labeling the protein to be purified, it is mixed with streptavidin agarose gel (such as Streptavidin Agarose/Sepharose), and the labeled protein will closely bind to streptavidin on the filler. By washing to remove impurities, and then washing the target protein under appropriate conditions (such as boiling SDS-PAGE buffer, 0.1% SDS treatment, or 95% formamide+10 mM EDTA treatment at pH 8.2), high-purity products can be obtained.

Taking the industrial production of recombinant human insulin as an example, high-quality streptavidin magnetic beads can bind more than 1200 pmol of free biotin per milligram, and the purified protein purity can reach over 95%.

3.2 Immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP)
The application of streptavidin magnetic beads in immunoprecipitation greatly improves experimental efficiency. Researchers can fix biotinylated antibodies onto streptavidin magnetic beads and co incubate them with cell lysate.

The antibodies specifically capture the target protein and its interacting proteins, forming a "magnetic bead antibody target protein" complex. It can be quickly separated under the action of an external magnetic field, with simple operation and high sensitivity. In ChIP experiments, streptavidin magnetic beads were also used to capture biotinylated DNA protein complexes and study the interaction between proteins and DNA.

3.3 Pull down experiment
In the study of protein-protein interactions, magnetic beads can capture biotinylated bait proteins and then "fish" for pre proteins that interact with them. For example, when studying key signaling pathways in tumor cells, specific antibodies that recognize P53 protein are immobilized on streptavidin magnetic beads and co incubated with tumor cell lysate to effectively enrich P53 and its associated proteins. Compared to traditional immunoprecipitation methods, magnetic bead IP has higher sensitivity and specificity, and can more effectively enrich low abundance target proteins.

3.4 RNA Pull-down
Magnetic beads are also suitable for RNA pull-down experiments. After binding with streptavidin magnetic beads, biotinylated RNA probes can capture proteins that interact with the RNA from cell lysates for studying RNA protein interaction networks.
3.5 Single stranded DNA preparation and nucleic acid extraction
It can bind with biotinylated oligonucleotides for the preparation of single stranded DNA and efficient extraction of nucleic acids, playing an important role in molecular cloning and genetic engineering.

4. High throughput detection and drug screening
4.1. TR-FRET testing platform
It can be coupled with luminescent chelates such as europium chelates to perform signal linkage and molecular capture in TR-FRET (time-resolved fluorescence resonance energy transfer) detection system. For example, THUNDER ™ The europium labeled streptavidin specifically designed for the TR-FRET experimental platform can stably bind to various biotinylated modified molecules and is widely used in protein interaction analysis and preliminary screening of target drugs.

This system is homogeneous and high-throughput, making it a powerful tool for modern drug development.

4.2 Targeted drug delivery
Its biotin binding properties make it an ideal carrier for drug targeted delivery systems. By coupling drug molecules with biotin and utilizing the targeted binding ability of streptavidin, precise drug delivery can be achieved, improving treatment efficiency and reducing side effects.

4.3 Biosensors and Biochips

It is the core material for the production of biosensors and biochips. By immobilizing streptavidin on the surface of the chip as a capture element, biotin labeled protein probes or antibodies can be immobilized on it, achieving high-throughput and high-sensitivity detection. For example, in early screening protein chips for cardiovascular disease, streptavidin magnetic beads are fixed on the chip substrate and used to bind biotinylated protein probes. This can accurately detect abnormalities in patients in the early stages of disease and when blood marker concentrations are as low as cTnI of 0.01 ng/mL, advancing the diagnostic window by 2-3 hours compared to traditional diagnostic methods.

5. Cell Biology and Imaging
5.1 Cell surface labeling and fluorescent cell sorting
Fluorescent labeled streptavidin (such as Vari Fluor 488 Streptavidin) is widely used for cell surface labeling and fluorescent cell sorting. Its emission spectrum covers the entire visible and near-infrared light spectrum, and in multi-color labeling experiments, it can easily enter the nucleus and cytoplasmic structure, label target proteins for imaging and analysis.

5.2 Nanotechnology and Material Self Assembly
Its tetravalent binding properties make it an ideal "bridge" for self-assembly of nanomaterials. Through the targeted binding of biotin streptavidin, various nanostructures can be constructed for interdisciplinary research such as biological imaging and environmental monitoring.

6. Anti streptavidin antibody
With the widespread use of the streptavidin biotin system, a often overlooked issue has emerged - endogenous anti streptavidin antibodies (ASA) in the sample may interfere with the detection system. Research has found that the binding of ASA to SA in the reaction system can affect the correct construction of specific antigen antibody systems, resulting in compromised accuracy of coupling, detection, or diagnostic results.

To address this challenge, anti streptavidin antibody detection kits have emerged, which can be used to detect endogenous ASA in patient samples and avoid its interference with the BAS system. Roche Custom Biotech has developed an inactive streptavidin mutant (Streptavidin rec. inactive, poly), which can be added to the current system without additional steps to specifically block and eliminate ASA interference, improving the accuracy and specificity of the detection system.

7. Biotin interference
Although the streptavidin biotin system is powerful, it faces an increasingly severe challenge - biotin interference.

When there is a high concentration of free biotin in the test sample, it competes with biotinylated antibodies for the binding site of streptavidin, resulting in distorted detection results.

Oral administration of 100 mg biotin can lead to a peak plasma concentration of 375-450 ng/mL; Patients with multiple sclerosis can take 300 mg/d orally, and their plasma biotin levels can exceed 1000 ng/mL; Daily intake of 500 mg of biotin can lead to extremely high concentrations of biotin in the blood. In sandwich immunoassays, excessive biotin can produce erroneous low results; In competitive immunoassays, it can lead to erroneous high results.

Numerous studies have shown that, TSH, Thyroxine PTH, Endocrine tests such as adrenocorticotropic hormone, prolactin, testosterone, cortisol, as well as tumor markers and cardiac functional markers, are all affected to varying degrees by biotin interference. The FDA has successively issued warning information and corresponding guidelines. The coping strategies include: serial dilution of samples, switching to non BAS methods such as liquid chromatography-mass spectrometry, retesting after biotin clearance (as short as 2 hours, as long as 15 days or even months), or using free biotin scavengers.