What is Biotin?
Biotin, with a molecular weight of 244.31, consists of two cyclic structures. The first ring is the imidazolone ring, the primary site for binding with avidin; the second ring is the thiazole ring, with a valeric acid side chain ending in a carboxyl group, which is the unique structure for binding with antibodies and other large biomolecules. By chemically modifying the carboxyl group of biotin, various derivatives with active groups, known as activated biotins, can be produced. These activated biotins can be optimized for specific purposes, such as solubility, arm length, and cleavability, to suit the needs of binding with various large biomolecules.
What is Biotinylation?
Biotinylation refers to the process of attaching a chemical substance or molecule to biotin. Biotin, also known as vitamin B7 or vitamin H, is a water-soluble vitamin that plays a crucial role in many biochemical processes in various organisms. Biotin can be synthesized chemically or obtained from natural sources, such as food. Biotinylation is commonly used in biological laboratories to label and detect specific molecules or proteins. In this process, biotin binds with the target molecule or protein, and the interaction can be detected or purified through the specific interaction between biotin and other molecules.
What is the Biotin-Streptavidin System?
Streptavidin is a protein with four identical binding sites that can form a very tight complex with biotin, representing one of the strongest known non-covalent interactions (dissociation constant, Kd, approximately 10x-15 mol/L). The bond between biotin and streptavidin is rapid and remains unaffected by extremes in pH, temperature, organic solvents, and other denaturing agents. The binding between biotin and streptavidin is highly specific and characterized by high affinity. This affinity makes the biotin-streptavidin complex a valuable tool for detecting, separating, and purifying many biological molecules, such as proteins, DNA, or RNA. The interaction between biotin and streptavidin is widely used in labeling and purification techniques in various biological experiments, including protein assays, Western blot analysis, immunohistochemistry, and other molecular biology experiments.
A major advantage of the biotin-streptavidin system is its ability to enhance detection sensitivity. This is largely attributed to the tetrameric conformation of streptavidin. A single streptavidin protein can bind with high affinity and selectivity to four biotin molecules, amplifying weak signals and increasing the detection sensitivity of low-abundance targets in mammalian cells or tissues through a simple workflow. Another key advantage is the versatility of the biotin-streptavidin system. As streptavidin can bind to various reporter labels, it can easily be incorporated into almost all immunoassays. For example, biotinylated enzymes are widely used in enzyme-linked immunosorbent assays (ELISA), while fluorescently labeled streptavidin, such as iFluor 488 streptavidin, is extensively used in cell surface labeling, fluorescence-activated cell sorting (FACS), and other fluorescence detection imaging applications.
Applications of Biotin Labeling
In many protein research applications, proteins labeled with biotin (biotinylation) are commonly detected or purified using streptavidin conjugates. This includes:
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Immunohistochemistry (IHC)
- Power Styramide signal amplification, an alternative to tyramide
- Western blot
- Immunofluorescence microscopy
- Cell surface labeling
- Affinity purification
- Fluorescence-Activated Cell Sorting (FACS)
- Flow cytometry
Featured Biotin Labeling Services at BOC Sciences
| Services | Description |
| Biotin Labeled Proteins | Using the characteristics of the biotin-affinity system, proteins can be biotinylated to bind specifically to affinity proteins for the purposes of antigen-antibody recognition detection, biomolecule capture, and protein interaction detection. |
| Biotin Labeled Oligonucleotides | Biotin-labeled oligonucleotides are compounds in which a biotin molecule is attached to an oligonucleotide molecule. |
| Biotin Labeled Nucleic Acids | Biotin-labeled nucleic acids can be used in in situ hybridization and immunohistochemistry to detect and localize the presence and distribution of target nucleic acid sequences in tissues or cells. |
| Biotin Labeled Peptides | Biotinylated peptides are compounds in which biotin is covalently bound to a peptide chain. |
| Biotin Labeled Magnetic Nanoparticles | Magnetic nanoparticles have a highly sensitive magnetic signal that can be manipulated and detected by an external magnetic field. By labeling biotin onto magnetic nanoparticles, this highly sensitive signal can be combined with the specific binding of biomolecules to achieve highly sensitive detection of low concentrations of target molecules. |
General Procedure for Biotin Labeling
- Ensure that the protein concentration of the target molecule is not too low. If the concentration is too low, methods such as ultrafiltration concentration should be employed to raise the concentration to at least 0.1 mg/mL.
- A protein can be labeled with multiple biotins. During labeling, there should be an appropriate ratio between biotin and the target molecule to ensure that the labeling does not affect the activity of the target molecule. The recommended biotin: antigen protein ratio is 1-3:1, and the biotin: antibody ratio is 5-20:1, or as per the recommended ratio in the labeling kit instructions.
- To reduce steric hindrance effects and meet requirements for detection sensitivity and specificity, a cross-linking arm structure can be introduced between biotin and the labeled molecule.
- If the coupling group is amino, the system of the target molecule must not contain free amines (Tris, amino acids), carrier proteins (BSA), or other interfering substances. If interference is present, repeatedly ultrafiltrate with the labeling buffer to ensure thorough removal. If other coupling agents are used, the target molecule system must not contain substances with corresponding free groups.
- If the target molecule is of relatively small molecular weight, pay attention to the molecular weight cutoff of ultrafiltration tubes, desalting columns, and dialysis bags during the purification process.
- The volume of the labeling reaction should be at least 100 μL to achieve the minimum loading volume during purification.
- After the labeling reaction (in the amino coupling system), an appropriate amount of small molecules containing free amino groups can be added to the system to neutralize free biotin activation reagents, improve purification efficiency, and reduce false positives and high backgrounds in subsequent reactions.
- The protein content of the labeled and purified protein can be determined using A280 or the BCA method.
- Labeled and purified proteins can be stored for an extended period according to the protein properties, reducing the number of freeze-thaw cycles.
Biotinylation of Nucleotides and Oligos
Biotin-modified nucleotide analogs, such as dUTP and dCTP, can be enzymatically incorporated into DNA or RNA fragments. This is commonly used in techniques such as Fluorescence In Situ Hybridization (FISH), DNA arrays, microarrays, and other hybridization technologies. Standard enzyme-catalyzed non-radioactive DNA labeling reactions include 3′ end labeling, cDNA labeling, nick translation, PCR, and random primer labeling. Each biotinylated nucleotide contains a spacer region of 11, 14, 16, or 20 atoms between biotin and its connection point on the nucleotide. For example, common ones like Biotin-dATP or Biotin-dCTP are used in Hi-C experiments at cleavage sites; biotin-labeled linkers (21bp dsDNA) can be used in ChIAPET experiments to connect two chromatin DNA fragments; in RNA pull-down experiments, biotin is incorporated into RNA probes or mRNA during in vitro transcription (using Biotin-16-UTP to replace UTP). The degree of biotin labeling for each RNA transcription product can be controlled by adjusting the ratio of NTP to Biotin-NTP in the transcription reaction. Some kits control the ratio of Biotin-16-UTP to approximately 35% to achieve a balance between reaction efficiency and labeling efficiency. Biotin-labeled RNA probes or mRNA obtained can be detected using fluorescence tags, enzymes, or streptavidin conjugated with antibodies (Streptavidin), applicable in RNA pull-down or other enrichment experiments. In reverse transcription reactions, biotin-modified primers are used to synthesize cDNA with biotin labels. Additionally, in RNA-seq library construction, biotin-labeled Oligo(dT) hybridizes with the poly A tail of mRNA at the 3′ end, and the resulting hybrid is washed and captured using streptavidin-coated paramagnetic beads to form a hybrid-bead complex, which is then captured using a magnetic rack.
How to Dissociate Biotinylated Molecules from Streptavidin Magnetic Beads?
The streptavidin-biotin interaction is known as one of the strongest non-covalent biological interactions between proteins and other molecules. The bond between biotin and streptavidin forms rapidly and remains unaffected by various extreme factors such as pH, temperature, organic solvents, and denaturing agents. When dissociating biotin from streptavidin-coated magnetic beads, if biotin derivatives or modified streptavidin are used in the experiment, specific and generally mild methods are required for dissociation. Otherwise, harsh methods are usually needed, and they may denature the streptavidin. Two methods are discussed below.
For biotinylated nucleic acids: To dissociate biotinylated nucleic acids from Dynabeads streptavidin-coated magnetic beads, the beads can be incubated in 95% formamide + 10 mM EDTA at pH 8.2, incubated at 65°C for 5 minutes, or at 90°C for 2 minutes. The beads can then be pulled to the side of the tube using a magnetic rack, and the supernatant containing the biotinylated nucleic acid can be collected from the tube. Some studies suggest that complete dissociation can be achieved at high temperatures (120°C, 15 minutes) in a saline solution. Additionally, dissociation can be achieved in a 6 mol/L HCl-guanidine hydrochloride solution (pH 1.5). It has been noted that biotin-labeled nucleic acid probes should not be extracted with phenol, as the biotinylated nucleic acids may enter the phenol layer and be lost. Holmberg et al. (Electrophoresis 26, 501-510, 2005) reported that a short incubation in deionized water could release biotinylated DNA from streptavidin-coated magnetic beads (not tested).
For biotinylated proteins: Biotinylated proteins can be boiled in 0.1% SDS or SDS-PAGE buffer for 3 minutes to dissociate them from the magnetic beads.
Binding Length and Capacity of Streptavidin Magnetic Beads
The binding capacity of streptavidin magnetic beads to DNA fragments depends on the size of the fragments due to steric hindrance. For example, a 500 bp fragment binds twice as much to Dynabeads M-280 Streptavidin as a 1,000 bp fragment. Longer DNA fragments occupy more space around the beads, making it more challenging for them to “find” the streptavidin on the beads. Shorter fragments can approach streptavidin more easily.
Salt concentration can influence the binding efficiency of biotinylated nucleic acids to Dynabeads Streptavidin magnetic beads. For biotinylated DNA fragments within 1 kb, the optimal binding conditions are 1 M NaCl (final concentration), incubation at 25℃ for 15 minutes; longer DNA fragments should be incubated overnight. Biotinylated antibodies should be fixed in PBS buffer containing 0.1% BSA at pH 7.4. As free biotin binds to Dynabeads Streptavidin magnetic beads faster than larger molecules, it is essential to ensure that the sample does not contain an excess of free biotin. HPLC or FPLC should be used to recover biotinylated oligonucleotides to avoid the presence of free biotin in the sample. Since the size of the specific molecules to be fixed and the biotinylation process will affect the binding capacity of the beads, a titration method can be used to optimize the bead usage in each individual application.
Are Dynabeads Streptavidin Magnetic Beads RNase-free?
Dynabeads Streptavidin Magnetic Beads are not provided in the form of an RNase-free solution. When working with RNA, the beads should be washed twice with a solution of 0.1 M NaOH/0.05 M NaCl treated with DEPC, each time for 1-3 minutes. DEPC is highly toxic but effective in removing RNase. After washing, the beads can be resuspended in a solution of 0.1 M NaCl treated with DEPC. “DEPC treatment” refers to the addition of 0.1% DEPC, mixing, and incubation at room temperature for 1 hour. Subsequently, the DEPC-treated solution should be autoclaved under high pressure to deactivate DEPC.