Frequently Asked Questions - Streptavidin-Coupled Dynabeads
Frequently asked questions
- Which Streptavidin-Coupled Dynabeads are best for my application?
- What do M-280, M-270 or MyOne mean?
- What is the Dynabeads kilobaseBINDER Kit?
- Can I purchase the Binding Solution in the Dynabeads kilobaseBINDER Kit separately?
- How many biotin binding-sites are there per streptavidin molecule?
- Which buffers are recommended for immobilizing biotinylated molecules?
- Which Streptavidin-Coupled Dynabeads do I use to isolate RNA/DNA binding proteins?
- How do I measure the binding of biotinylated molecules on streptavidin beads?
- Can the Streptavidin-Coupled Dynabeads be used directly in a PCR reaction?
- I have a dsDNA biotinylated on the beads. How can I dissociate the non-biotinylated DNA strand from the biotinylated one?
- How do I dissociate my biotinylated molecule from Streptavidin-Coupled Dynabeads?
- Are the Streptavidin-Coupled Dynabeads reusable?
- I am using Dynabeads M-280 Streptavidin to isolate DNA binding proteins and added my biotinylated oligo to my protein sample first. I am afraid the B&W buffer suggested for coating of oligos to the beads could break my DNA-protein interaction?
- How do I elute my target protein from biotinylated DNA?
- How do I elute my target from biotinylated antibody without eluting the antibody off the Dynabeads?
- As a negative control I mixed my uncoated Streptavidin-Coupled Dynabeads with my sample but got lots of non-specific binding to the beads. Why?
- Can streptavidin leak from the Streptavidin-Coupled Dynabeads?
This will depend on the properties of your sample, the buffers and solutions used and your downstream application. For an overview, see Invitrogen Streptavidin-Coupled Dynabeads. In general, they can all be used, but some may perform better than others in particular applications due to their characteristics. Invitrogen Dynabeads M-280 Streptavidin and Invitrogen Dynabeads MyOne Streptavidin T1 are used for both protein and nucleic acids applications. Invitrogen Dynabeads M-270 Streptavidin and Invitrogen Dynabeads MyOne Streptavidin C1 are preferred for molecular diagnostics and for handling samples with high concentration of chaotropic salt, as well as in immunoassays involving small biotinylated antigens and in applications that are not compatible with BSA as these beads are not blocked with BSA. The Dynabeads Streptavidin Trial Kit allows you to try 1 mL of all four (while supplies last). The Dynabeads MyOne Streptavidin C1/T1 offer an increased binding capacity and slower sedimentation rate, making them ideal for automated applications and/or for when larger amount of biotinylated molecules or their specific target need to be isolated.
'M' stands for 'magnetic' and 280 /270 indicates the diameter of the beads. Both are 2.8 micron. M-280 denotes the hydrophobic 2.8 micron beads, while M-270 denotes the hydrophilic 2.8 micron beads. MyOne beads are 1 micron in diameter.
The Invitrogen Dynabeads kilobaseBINDER Kit is designed to bind large (>2 kb) biotinylated DNA or RNA fragments. The kit contains Dynabeads M-280 Streptavidin plus a unique, patented Binding Solution. The Binding Solution enables efficient capture of long fragments of biotinylated DNA as well as small biotinylated probe hybridized with long non-biotinylated DNA.
Currently, this buffer is only sold as part of the kit and is not available separately
Streptavidin is a protein composed of four identical subunits, each containing a high affinity binding site for biotin (KD = 10-15 M). Streptavidin has the same biotin binding properties as avidin, but it has a low isoelectric point (pI=5) and no carbohydrate groups, resulting in low nonspecific binding.
PBS is the recommended immobilization buffer for biotinylated proteins or other molecules. For immobilization of biotinylated nucleic acids, we recommend the following Binding and Wash (B&W): 10.0 mM Tris-HCl (pH 7.5) 1.0 mM EDTA 2.0 M NaCl.
Dynabeads M-280 Streptavidin has been extensively used for this application so this is the product we recommend.
Assay the supernatant for unbound molecules. This will determine the amount of molecule bound to the Dynabeads. For nucleic acids, the concentration can be checked by OD-readings, or by running a gel. For proteins, the concentration in the supernatant can be determined by a spectrometer using a protein assay like BCA. Alternatively you can label the molecule with radioactivity or fluorescence and measure the concentration of molecule directly on the beads (former) or in the supernatant (latter).
Yes, the beads can be used directly in PCR, as they do not inhibit enzymatic reaction. Slight PCR-inhibition has been seen when 75–100 µg of Dynabeads M-280 Streptavidin was used in 50 µL PCR reaction, and a concentration over 100 µg per 50 µL reaction seems to inhibit PCR completely. Dynabeads M-270 Streptavidin do not show any inhibitory effect on PCR at these concentrations.
I have a dsDNA biotinylated on the beads. How can I dissociate the non-biotinylated DNA strand from the biotinylated one?
There are two methods to dissociate the non-biotinylated DNA from the biotinylated DNA strand. The following protocols are based on using 20 µL of Dynabeads Streptavidin, but are scalable. Both methods may release very small amounts of complementary biotinylated strand from streptavidin. If it is critical that no biotinylated strand is released, either adopt a different biotin modification using dual biotin (two biotin groups in sequence) or covalently bind DNA to e.g., Dynabeads M-270 Carboxylic Acid.
- Wash the DNA coated Dynabeads in 50 µL 1 x SSC.*
- Resuspend the beads in another 50 µL of 1 x SSC Incubate at 95 °C for 5 minutes.
- Quickly put the tube in magnet stand for 1–2 minutes and transfer the supernatant to a new tube.
- The supernatant contains your non-biotinylated DNA strand.
- Wash the DNA coated Dynabeads in 50 µL 1 x SSC.*
- Resuspend the beads in 20 µl of freshly prepared 0.15 M NaOH.
- Incubate at room temperature for 10 minutes. Put the tube in magnet stand for 1–2 minutes and transfer the supernatant to a new tube.
- The supernatant contains your non-biotinylated DNA strand. Neutralize the probe by adding 2.2 µL 10 x TE, pH 7.5 and 1.3 µL 1.25 M acetic acid.
- Wash the Dynabeads coated with biotinylated strand once with 50 µL 0.1M NaOH, once with 50 µL of B&W buffer and once with 50 µL TE buffer.
*1 x SSC (0.15 M NaCl, 0.015 M sodium citrate. Dissolve the reagents in 800 mL water. Adjust pH to 7.0 with NaOH. Adjust the volume to 1 liter with water).
The streptavidin-biotin interaction is the strongest known non-covalent, biological interaction between a protein and molecule. The bond formation between biotin and avidin is very rapid and, once formed, is unaffected by wide extremes of pH, temperature, organic solvents and other denaturing agents. Unless derivative forms of biotin or modified streptavidin have been adopted for your experiment, requiring a specific form and normally gentle way to dissociate biotin from streptavidin, often very harsh methods are required to dissociate the biotin from streptavidin which will denature the streptavidin. A couple of these methods are discussed below. See also Elution Guidelines.
Biotinylated nucleic acids:
To dissociate biotinylated nucleic acids from Streptavidin-Coupled Dynabeads, incubate the beads in 95% formamide + 10mM EDTA, pH 8.2 for 5 minutes at 65°C or for 2 minutes at 90°C. Pull the beads to the tube wall with the magnet and remove the supernatant containing the biotinylated nucleic acid from the tube. Holmberg et al. (Electrophoresis 2005, 26, 501–510), report release of biotinylated DNA from streptavidin beads after short incubation in de-ionized H2O but this method has not been tested by our R&D department.
For biotinylated proteins, boil the beads in 0.1% SDS or SDS-PHAGE buffer for 3 min.
For most applications involving dissociation of biotinylated molecules from streptavidin it is not possible to reuse the beads. The streptavidin-biotin bond is one of the strongest biological bonds known, and the conditions necessary to break this bond also destroy the streptavidin. However, if the Dynabeads Streptavidin have been used in applications such as isolation of DNA binding proteins and release of proteins from DNA is done by gentle methods like using high salt buffers that do not interfere with biotin-streptavidin interaction, the beads with immobilized probe may be reused.
I am using Dynabeads M-280 Streptavidin to isolate DNA binding proteins and added my biotinylated oligo to my protein sample first. I am afraid the B&W buffer could break my DNA-protein interaction?
This buffer is intended for immobilizing oligo on to the beads in the absence of protein or when the direct method is applied. For the indirect method, when the oligo is first mixed with the protein sample and Dynabeads M-280 Streptavidin are added to capture DNA-protein complex, a buffer with salt concentration as low as 150 mM should be applied. The most common methods are to use either a high salt buffer or boil the Dynabeads with DNA-protein complex in SDS sample buffer for 3–4 minutes. With a high salt buffer, a salt concentration higher than 1 M is normally applied to break DNA-protein interaction. The exact amount of salt required depends on the affinity of the protein for oligonucleotides and should be determined for each application.
You need to use mild elution methods like buffer with high salt (>1 M salt) or low pH. Low pH elution buffers such as 0.1 M glycine•HCl, pH 2.5–3.0 are effective for most antibody-antigen interactions. Note that boiling in SDS will also elute the antibody.
As a negative control I mixed my uncoated Streptavidin-Coupled Dynabeads with my sample but got lots of non-specific binding to the beads. Why?
When exposed to a sample consisting of different types of molecules, any solid phase matrix can interact with these molecules due to hydrophobicity, charge or other types of interactions. It is not surprising that you get non-specific binding to the beads. This method is actually used for pre-clearing of sample and is not considered a good negative control. When pre-blocked and coated with a specific molecule, beads show a lot less non-specific binding than when they are not coated. As a negative control, you could try beads that are coated with an irrelevant molecule.
Can streptavidin leak from the Streptavidin-Coupled Dynabeads?
The streptavidin molecule is covalently attached to the bead's surface. However, not all of the four streptavidin subunits are covalently coupled to the beads, typically only one or two. Streptavidin is like other proteins; if heated it can denature and dissociate into subunits. If streptavidin-coupled Dynabeads are boiled, some of the streptavidin subunits may be released (as monomers or aggregates) from the beads. The covalently bound streptavidin subunits will not be affected by such treatment. When streptavidin is bound to biotin, the streptavidin-biotin complex is more stable than the streptavidin itself. Under normal, recommended conditions, only negligible leakage of streptavidin from the beads is detected (less than 0.2% of total attached streptavidin after 2 months at 37°C).