Streptavidin-Biotin System
Streptavidin is a tetrameric biotin-binding protein that is isolated from Streptomyces avidinii and has a mass of 60,000 daltons. One molecule of streptavidin has the ability to bind up to four biotin molecules (Figure 1), making this interaction ideal for both purification and detection strategies. The dissociation constant (Kd) for this interaction is approximately 10-15 M. This tight and specific binding occurs very rapidly and can withstand extreme pH values, temperatures, organic solvents, and other denaturing agents.
Streptavidin has an isoelectric point of approximately 6.0, and its moderate overall charge reduces electrostatic interactions with other molecules, thereby decreasing nonspecific binding. These advantages make the streptavidin-biotin system superior to the avidin-biotin system in practical applications, having less nonspecific adsorption, lower background, and better signal-to-noise ratio. This makes Streptavidin an ideal reagent choice for many detections systems.
Applications for which the streptavidin -biotin interaction is used include:
Application 1: Immunoprecipitation
Streptavidin can be conjugated to various carriers such as magnetic beads and agarose matrices, becoming a highly specific affinity medium for capturing various biotin-labeled ligands. MCE Streptavidin Magnetic Beads have been cited in 85+ publications covering various applications, such as IP, ChIP, RNA pull-down, protein purification, and more.
Case 1: Immunoprecipitation (IP)
To further confirm whether Rab11a played a role in MSC-sEV recycling under hypoxia, we used streptavidin beads to coprecipitate biotin-labeled MSC-sEVs and their interacting proteins in NPCs while the negative control group was treated with unlabeled MSC-sEVs. ALIX, a commonly used sEV marker, was used to detect coimmunoprecipitated MSC-sEVs. Interestingly, we found that Rab11a was coprecipitated by streptavidin beads in both normoxia and hypoxia groups but not in biotinylated MSC-sEV or NPCs + unlabeled MSC-sEV groups, which indicated an enhanced interaction between Rab11a and internalized MSC-sEVs under hypoxia (Figure 2)[1].
Case 2: Chromatin Immunoprecipitation (ChIP)
The researchers hypothesize that CnHsf3 can be activated through direct oxidation of its DNA binding domain (DBD). A protein–DNA co-IP experiment was carried out using three biotinlabeled CnHsf3-mitochondrially targeted oligonucleotides (Figure 3). Results showed that NaClO-treated CnHsf3 DBDs readily bind to all target oligonucleotides, whereas the untreated CnHsf3 DBD remained unbound or demonstrated limited binding as compared to the NaClO-treated sample. The EMSA analysis demonstrated that the NaClO-treated DBD shifted to give rise to a high molecular weight DNA–protein complex. These indicated that the function of CnHsf3 is related to the oxidation of its DBD [2].
Case 3: RNA Immunoprecipitation (RIP)
The researchers hypothesize that YTHDC1 is involved in regulating the expression of SQSTM1 in HaCaT cells. First, tests were performed to determine whether YTHDC1 protein interacted with SQSTM1 mRNA. RNA Immunoprecipitation (RIP)-qPCR experiments revealed that the YTHDC1 protein could interact with SQSTM1 mRNA. To further confirm this result, RNA affinity isolation was conducted, which verified that biotinylated SQSTM1 mRNA interacted with the YTHDC1 protein [3].
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Tips: How to dissociate biotin molecules from streptavidin magnetic beads?
Here are two example methods:
1)?Dissociation of biotinylated nucleic acids: To separate biotinylated nucleic acids from streptavidin magnetic beads, incubate the beads in 95% formamide + 10 mM EDTA at pH 8.2, for 5 minutes at 65°C or 2 minutes at 90°C.
2)?Dissociation of biotinylated proteins: boiling the beads in 0.1% SDS or SDS-PAGE buffer for 3 minutes.
Application 2: Signal Amplification in Imaging
The noncovalent, high affinity of biotin for streptavidin, with four biotin-binding sites per streptavidin molecule, allows more signal to be concentrated at a detection site. An optimized biotin-to-probe ratio can greatly increase the signal output of a detection system making it possible to create very sensitive assays for low to medium abundance targets in cells or tissues. Streptavidin can be conjugated with various fluorescent dyes or reporter labels, while biotinylated antibodies and enzymes have high labeling rates without affecting protein activity. Consequently, the streptavidin-biotin system is applicable in nearly all immunoassay experiments. These advantages render streptavidin-biotin labeling technology more sensitive than conventional enzyme-linked immunoassays, radioimmunoassays, and fluorescence immunoassays, thereby facilitating the detection of trace amounts of antigens and antibodies.
Fluorescent streptavidin conjugates, such as Vari Fluor 488-Streptavidin, is widely used in cell surface labeling, fluorescence-activated cell sorting (FACS), and other fluorescence detection imaging applications.
MCE provides a variety of Vari Fluor fluorescent streptavidin conjugates (Table 1). The emission spectra of these Vari Fluor dyes encompass both the visible and near-infrared light ranges. In multi-color labeling experiments, these dyes can readily penetrate cells to label target proteins, facilitating imaging and analysis.