Dynabeads™ MyOne™ Streptavidin T1, 10 mL - FAQs

We do not recommend this as streptavidin becomes hydrophobic and aggregates during denaturation.

Which product to choose depends on the properties of your sample, the buffers and solutions applied, as well as the downstream application. In general, all Dynabeads Streptavidin beads can be used in applications involving biotinylated ligands; however, some beads may perform better than others in particular applications due to their characteristics. Dynabeads M-280 Streptavidin beads and Dynabeads MyOne Streptavidin T1 beads are commonly used for protein and nucleic acid applications. Dynabeads M-270 Streptavidin beads and MyOne Streptavidin C1 beads are preferred for nucleic acid diagnostics, specifically with samples that have a high concentration of chaotropic salts, immunoassays involving small biotinylated antigens, and in applications that are not compatible with BSA, as these beads are not blocked with BSA. Dynabeads MyOne Streptavidin beads offer increased binding capacity and slower sedimentation rate, making them ideal for automated applications and when larger amounts of a biotinylated compound or its specific target need to be isolated. Please see the selection guide here ( https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html?icid=fr-strep-1).

You can assay the supernatant for unbound nucleic acid to determine the amount of nucleic acid bound to the Dynabeads Streptavidin beads. The concentration of nucleic acids can be checked by measuring the OD or by running them on a gel. Alternatively, the nucleic acids can be labeled radioactively and the concentration measured directly on the beads, or fluorescently and measured in the supernatant.

Our Dynabeads Streptavidin magnetic beads can be used directly in PCR or real-time PCR. However, you would have to empirically optimize the amount of beads to be used per volume of reaction.

The exact number of streptavidin molecules bound per bead is not measured, but is approximately 14-16 µg streptavidin per milligram Dynabeads M-280 Streptavidin magnetic beads.

All biotin reagents should contain a spacer arm, at least a 6-carbon linker, to reduce steric hindrance. This is because the bicyclic ring of biotin goes deep into the biotin binding cleft in streptavidin. A 6-carbon arm is the minimum length between biotin and the first base of sequence that is required to reduce the steric hindrance effect. The longer this distance is, the less the steric hindrance. A 6-carbon linker is standard linker size from most companies and should be enough for most applications. We recommend specific biotinylation at the 5'-end of the probe.

In direct capture, the biotinylated probe/ligand is first coupled to the Dynabeads magnetic beads followed by addition of your sample. In indirect capture, the biotinylated probe/ligand is first added to the sample followed by addition of your Dynabeads magnetic beads. Precoupled ligand for direct capture allows you to reuse the Dynabeads magnetic beads, while an indirect approach can be beneficial when the concentration of your target is low, specific affinity is weak, or the binding kinetics is slow. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/nucleic-acid-capture-assays.html) for a diagram of the capture.

- One milligram of Dynabeads M-280 Streptavidin magnetic beads typically binds 650-900 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 µg of biotinylated antibody, 10 µg of biotinylated double-stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.

- One milligram of Dynabeads M-270 Streptavidin magnetic beads typically binds more than 950 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 µg of biotinylated antibody, 10 µg of biotinylated double- stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.

- One milligram of Dynabeads MyOne Streptavidin C1 magnetic beads typically binds more than 2,500 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 µg of biotinylated antibody, 20 µg of biotinylated double-stranded DNA, or 500 pmol of biotinylated single-stranded oligonucleotides.

-One milligram of Dynabeads MyOne Streptavidin T1 magnetic beads typically binds 1,100-1,700 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 µg of biotinylated antibody, 20 µg of biotinylated double- stranded DNA, or 400 pmol of biotinylated single-stranded oligonucleotides.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

This is dependent on coating or the biotinylated molecule properties. Our recommendation is that this should be tested to find optimum conditions for the specific assay.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

The density of Dynabeads magnetic beads is a challenging property to determine. The reason is that Dynabeads magnetic beads have a 17-37% magnetic iron oxide content in order to have a reasonable magnetic separation time, and the density of the iron oxide is about 4.9 g/cm³. Dynabeads magnetic beads are composite materials, being a mix of polymers and iron oxide, and there are very few polymers that have a density below 1.

The sedimentation rate depends on the bead diameter squared, so the sedimentation of a 1 µm bead is much slower than that of 2.8 µm. The effect of diameter on sedimentation rate is to some extent counteracted by the fact that smaller beads need to have a higher content of iron oxide for magnetic separation applications. Typically, our M-280 Dynabeads (diameter 2.8 µm) have a density of 1.4 g DS/cm³ (DS = dry substance), our M-270 Dynabeads (diameter 2.8 µm) and M-450 Dynabeads (diameter 4.5 µm) have a density of 1.6 g DS/cm³, and our MyOne Dynabeads (diameter 1 µm) have a density of 1.8 g DS/cm³.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Dynabeads Cell Isolation and Expansion Support Center and Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Several methods can be used.

Using heat:
Wash the DNA-coated Dynabeads magnetic beads in 50µL 1x SSC. (To make SSC, dissolve 0.15 M NaCl, 0.015 M sodium citrate in 800 mL water. Adjust pH to 7.0 with NaOH. Adjust the volume to 1 L with water.)

Resuspend the beads in another 50µL of 1x SSC.

Incubate at 95 degrees C for 5 min.

Quickly put the tube in a magnet stand for 1-2 min and transfer the supernatant to a new tube. The supernatant contains the nonbiotinylated DNA strand.

Generally, heat destabilizes the interaction between biotin and streptavidin and can increase the release of biotinylated ligands from streptavidin. This effect varies in different reagents. In water, normally this effect is minimal, especially if it contains salt.

Using NaOH:< br />
Wash the DNA-coated Dynabeads magnetic beads in 50µL 1 x SSC.

Resuspend the beads in 20 µl of freshly prepared 0.15 M NaOH.

Incubate at room temperature for 10 min.

Put the tube in a magnet stand for 1-2 min and transfer the supernatant to a new tube. The supernatant contains the nonbiotinylated DNA strand.

Neutralize the probe by adding 2.2µL 10 x TE, pH 7.5, and 1.3µL 1.25 M acetic acid. The Dynabeads magnetic beads coated with a biotinylated DNA strand can be washed once with 50µL 0.1 M NaOH, once with 50µL of Binding and Wash buffer, and once with 50µL TE buffer.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

A probe length between 20 and 50 bases is generally okay. The capacity, measured as number of targets captured, is determined by the length of the targets and not as much by the density of probes on the surface.

If binding capacity is of importance, Dynabeads MyOne Streptavidin C1 magnetic beads is a good choice.

After biotinylated DNA has been bound to Dynabeads Streptavidin magnetic beads, the complex can be washed in water without influencing the streptavidin or the binding.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

Maintaining a high pH will maintain the negative charge on both the nucleic acid and the beads. So, we recommend keeping the pH high in combination with a low salt concentration. Additionally, to reduce the non-specific binding or high background, we recommend blocking the beads with BSA or BSA + Tween-20.

Separation of two DNA strands can be done by treating with either alkali or high temperature.

Using alkali, the nonbiotinylated strand can be eluted with 0.1 M NaOH. This treatment should normally not have any affect on the beads.

Wash the Dynabeads-DNA complex once in 2x Binding and Wash buffer prior to NaOH treatment and remove the supernatant. The high salt concentration will help to reduce the charge and hence minimize nonspecific binding.

Add freshly made (this is critical) 0.1 M NaOH to the Dynabeads-DNA complex and incubate at room temperature for 2-3 min (maximum 5 min) with rotation. Remove the supernatant containing the nonbiotinylated strand.

Wash the Dynabeads-DNA complex once more with 0.1 M NaOH and remove the supernatant. Most of the nonbiotinylated DNA will come off during the first elution.

Heating at 95 degrees C for 5 min in water is an alternative to the alkali treatment. This requires fast separation to prevent reannealing, preferably on ice. Please note that heating will cause some percentage of biotinylated DNA to be dissociated from streptavidin. Therefore, we usually recommend alkali treatment.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

Biotinylated and radioactively labelled antibodies can be used to estimate binding capacity.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

We do not recommend to re-use the Dynabeads since we can not be sure if the functional groups or the background have been removed. However, if the Dynabeads Streptavidin magnetic beads have been used in applications such as isolation of DNA binding proteins or hybridization capture of specific DNA sequences, the beads with immobilized probe may be reused up to ten times. However, for most applications, 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 molecules.

Storage should be at 2-8 degrees C. Freezing or drying of Dynabeads Streptavidin magnetic beads is not recommended. Provided the beads are stored correctly, quality is guaranteed until the expiration date stated on the label.

The beads should be washed twice, and then stored at 4 degrees C in the buffer they are supplied in. NaN3 can be added to the buffer as a preservative if the beads are to be stored for a longer period. It is also possible to store the beads in TE buffer, pH 8.0.

Breaking the biotin-streptavidin bond requires harsh conditions. For dissociating biotinylated DNA from Dynabeads Streptavidin magnetic beads, we recommend heating in 10 mM EDTA, 95% formamide, pH 8.2, for 5 min at 65 degrees C or for 2 min at 90 degrees C. Alternatively, the Dynabeads-DNA complex may be boiled for 5 min in 0.1% SDS.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

Dynabeads Streptavidin magnetic beads are not supplied in RNase-free solution. For RNA manipulations, the beads should be washed twice for 1-3 min in a DEPC-treated 0.1 M NaOH, 0.05 M NaCl solution. DEPC is very toxic and is used to get rid of RNases. After washing, the beads can be resuspended in a DEPC-treated 0.1 M NaCl solution. (DEPC treated means adding 0.1% DEPC to the NaCl solution, mixing, incubating for 1 hr at room temperature and autoclaving the DEPC-treated solution to destroy the DEPC).

Biotinylation easily inhibits enzymatic activities.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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 are covalently coupled. 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 degrees C).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Streptavidin is a protein made up 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 less nonspecific binding is observed. After immobilization on the beads, there are 2-3 binding sites free for interaction with biotin.

Find additional tips, troubleshooting help, and resources within ourDynabeads Nucleic Acid Purification Support Center as well as ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

The Dynabeads KilobaseBINDER Buffer is a viscous solution and therefore the beads will behave differently in this buffer than in other buffers. Be patient while resuspending the beads in the buffer and try to avoid pumping air into the tube. Flicking the tube containing the beads and buffer must be done very carefully.

If still not completely dissolved, leave it on a roller at 4 degrees C.

Dynabeads M-280 Streptavidin magnetic beads are supplied at 10 mg (approx. 6.7 x 10e8) beads per mL, dissolved in phosphate buffered saline (PBS), pH 7.4, containing 0.1% BSA and 0.02% NaN3 as a preservative.

Dynabeads M-270 Streptavidin magnetic beads are supplied at both 10 mg (approx. 6.7 x 10e8) beads per mL and at 50 mg (approx. 3.2 x 10e9) beads per mL. Both products are dissolved in PBS, pH 7.4, containing 0.02% NaN3 as a preservative.

Dynabeads MyOne Streptavidin C1 magnetic beads are supplied at 10 mg (approx. 7-12 x 10e9) beads per mL, dissolved in PBS, pH 7.4, containing 0.01% Tween 20 detergent and 0.09% NaN3 as a preservative.

Dynabeads MyOne Streptavidin T1 magnetic beads are supplied at 10 mg (approx. 7-12 x 10e9) beads per mL, dissolved in PBS, pH 7.4, containing 0.01% Tween 20 detergent and 0.02% NaN3 as a preservative.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Dynabeads M-270 Streptavidin magnetic beads and Dynabeads MyOne C1 magnetic beads have a negatively charged surface. The surface charge of the beads may in some samples cause the beads to float or become sticky or aggregate. The stickiness may be due to electrostatic interactions between the beads or between the beads and the tube wall. Usually we recommend washing the beads in a nonionic detergent like Tween 20 detergent before doing the experiment. The problem is usually reduced or eliminated by simply adding Tween 20 detergent to a final concentration of up to 0.1% to the beads, followed by resuspension and washing in buffer without the detergent. An incubation in the Tween 20 solution may be needed, e.g. 5-10 min at room temperature on a roller. In addition, we recommend using siliconized tubes. This treatment will most likely reduce the electrostatic potential of the beads.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Breaking the biotin-streptavidin bond requires harsh conditions. For dissociating biotinylated DNA from Dynabeads Streptavidin Beads, heat in 10 mM EDTA, pH 8.2, 95% formamide for 5 min at 65 degrees C or for 2 min at 90 degrees C. Alternatively, the Dynabeads-DNA complex may be boiled for 5 min in 0.1% SDS. Please be aware that these beads cannot be reused after this treatment.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

The capacity is dependent on the fragment size due to steric hindrance. For example, twice as many 500 bp fragments will bind to Dynabeads M-280 Streptavidin magnetic beads than a 1,000 bp fragment. Long DNA fragments will occupy more space around the beads and make it more difficult to "find" the streptavidin on the beads. Smaller fragments will access the streptavidin more easily. For DNA fragments greater than 2 kb, the Dynabeads kilobaseBINDER Kit is recommended. This kit contains Dynabeads M-280 Streptavidin magnetic beads and a special binding solution that enhances immobilization of long (greater than 2 kb) biotinylated DNA fragments. For DNA fragments greater than 1-2 kb, the Dynabeads kilobaseBINDER Kit is recommended, as the binding solution will enhance binding capacity. The binding solution will linearize the DNA so that it stretches out and the bases stack in a rigid structure (it will not work for shorter fragments such as plasmids or circular nucleic acids).

The salt concentration influences the efficiency of binding of biotinylated nucleic acids to the streptavidin-coupled Dynabeads magnetic beads. Optimal binding conditions for biotinylated DNA fragments (up to 1 kb) are achieved at 1 M NaCl (final concentration), 25 degrees C, and 15 min incubation time. Longer DNA fragments should be immobilized overnight. Biotinylated antibodies should be immobilized in PBS buffer, pH 7.4, supplemented with 0.1% BSA. Ensure that your sample does not contain excess free biotin, as the free biotin will bind Dynabeads Streptavidin Beads much more rapidly than larger molecules. Biotinylated oligonucleotides should be recovered by reverse phase HPLC or FPLC to avoid free biotin from being present in the sample. Titration is performed to optimize the quantity of beads used for each individual application, since both the size of the specific molecule to be immobilized and the biotinylation procedures will affect the binding capacity of the beads.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Storage should be at 2-8 degrees C. Freezing Dynabeads magnetic beads is not recommended. Provided the Dynabeads magnetic beads are stored correctly, quality is guaranteed until the expiration date stated on the label.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Dynabeads magnetic beads coated with antibody/ligand may be stored at 2-8 degrees C without loss of antigen binding capacity. For long-term storage, a final concentration of 0.02% NaN3 may be added to the antibody-coupled beads in a physiological buffer. Please note that not all coupled antibodies retain their function in long term storage. Verify your coupled antibody stability by testing in small scale. After storage, coated Dynabeads magnetic beads should be washed once in PBS/BSA for 5 min before use.

Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Standard elution methods are used to dissociate the isolated protein from Dynabeads magnetic beads. The most suitable elution method depends on the characteristics of the isolated protein and the desired downstream application of the eluted protein. In many cases, Dynabeads magnetic beads can be recovered for reuse after elution. Some standard elution methods are listed below.

-pH change: elution can be achieved by reducing pH (for example, by using 0.1 M citrate (pH 2-3) as the elution buffer). Dynabeads magnetic beads are stable between pH 4-13. If the Dynabeads magnetic beads are exposed for a prolonged period of time to pH below 4, the beads may be adversely affected.

-Change of ionic strength: high-salt concentration buffers (e.g., NaI, KI, MgCl, KCl) can be used to elute isolated proteins. Optimization is required, by step-wise elution starting at 1 M and increasing to 3 M.

-Affinity elution: with this method, the eluting agent competes for the binding of the protein or the binding of the ligand e.g., elution of glycoproteins from a lectin coupled to Dynabeads magnetic beads may be achieved by the addition of the free sugar.

-Denaturing eluents: as a last resort, denaturing eluents such as chaotropic salts may be used to alter the structure of the protein. The proteins on the bead surface and the eluted proteins will be denatured.

-Polarity reducing agents : substances that reduce the polarity of the buffer often disrupt the hydrophobic interactions between antibody and protein. Dioxane or ethylene glycol may be used to reduce the polarity of the eluent.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Storage should be at 2 to 8 degrees C. Freezing or drying of the Dynabeads magnetic beads is not recommended. Provided the Dynabeads magnetic beads are stored correctly, quality is guaranteed until the expiry date stated on the label.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The binding capacity of streptavidin-coupled Dynabeads magnetic beads is fragment length-dependent. Reduced binding capacity for large DNA fragments may be due to steric hindrance. For large DNA fragments (greater than 2 kb in size), we recommend using Dynabeads kilobaseBINDER Kit.

-Salt concentration affects the binding efficiency of biotinylated nucleic acids to the Streptavidin-coupled Dynabeads magnetic beads. Optimal binding conditions for biotinylated DNA fragments (up to 1 kb) are achieved at 1 M NaCl (final concentration), 25 degrees C and 15 min incubation time. Longer DNA fragments should be immobilized overnight. Biotinylated antibodies should be immobilized in PBS buffer pH 7.4, supplemented with 0.1% BSA.

-Ensure that your sample does not contain an excess of free biotin, as the free biotin will bind Streptavidin-coupled Dynabeads magnetic beads much more rapidly than larger biotinylated molecules. Biotinylated oligonucleotides should be recovered by reverse phase HPLC or FPLC to remove free biotin from the sample.

-We also recommend a titration to optimize the quantity of beads used for each individual application, since both the size of the specific molecule to be immobilized and the biotinylation procedures affect the binding capacity of the beads.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The streptavidin-biotin interaction is the strongest known non-covalent biological interaction between a protein and a ligand. Binding of biotin and streptavidin is very rapid and, once formed, the complex is unaffected by wide extremes of pH, temperature, organic solvents, and other denaturing agents. Normally, very harsh methods are required to dissociate the biotin from streptavidin, which will be irreversibly denatured by the procedure.

To dissociate biotinylated proteins from streptavidin, boil the beads in 0.1% SDS or SDS-PAGE buffer for 3 mins or incubate them in 8 M guanidinium hydrochloride, pH 1.5.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

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.

Using heat:

- 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 degrees C for 5 mins.
- Quickly put the tube in magnet stand for 1-2 mins and transfer the supernatant to a new tube.
- The supernatant contains your non-biotinylated DNA strand.

Using NaOH:

- 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 mins. Put the tube in magnet stand for 1-2 mins 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).

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

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).

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.

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) buffer: 10.0 mM Tris-HCl (pH 7.5) 1.0 mM EDTA 2.0 M NaCl.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Streptavidin is a protein composed of four identical subunits, each containing a high affinity binding site for biotin (K-D = 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 non-specific binding.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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 (https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html). 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 (https://www.thermofisher.com/order/catalog/product/11205D#/11205D) and Invitrogen Dynabeads MyOne Streptavidin T1 (https://www.thermofisher.com/order/catalog/product/65601#/65601) are used for both protein and nucleic acids applications.
- Invitrogen Dynabeads M-270 Streptavidin (https://www.thermofisher.com/order/catalog/product/65305#/65305) and Invitrogen Dynabeads MyOne Streptavidin C1 (https://www.thermofisher.com/order/catalog/product/65001#/65001) 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 (https://www.thermofisher.com/order/catalog/product/65801D#/65801D) 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.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Use mild elution conditions, e.g., a buffer with high salt or low pH. Heating the beads at 95 degrees C for 5 mins in SDS sample buffer will elute the antibody as well.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The streptavidin-biotin interaction is the strongest known non-covalent biological interaction between a protein and a ligand. Binding of biotin and streptavidin is very rapid and, once formed, the complex is unaffected by wide extremes of pH, temperature, organic solvents, and other denaturing agents. Normally, very harsh methods are required to dissociate the biotin from streptavidin, which will be irreversibly denatured by the procedure. Biotin-streptavidin interactions are more easily reversible when biotin derivatives with lower binding affinity are used along with chemically modified streptavidins with lower biotin binding affinity. When modified biotins and streptavidins are used, gentle methods for inducing reversible binding are available

To dissociate biotinylated proteins from streptavidin, boil the beads in 0.1% SDS or SDS-PAGE buffer for 3 mins or incubate them in 8 M guanidinium hydrochloride, pH 1.5.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

We do not recommend this because streptavidin becomes hydrophobic and aggregates during heat-induced denaturation.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The product to choose depends on the properties of your sample, the buffers and solutions you will use, as well as your downstream application. In general, all the Dynabeads Streptavidin beads can be used in applications involving biotinylated ligands. However, some beads may perform better than the others in particular applications due to their characteristics. Please refer to the Dynabeads Streptavidin Selection Guide (https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html#selection) for detailed information.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The streptavidin molecule is covalently attached to the surface of the beads; under normal, recommended conditions, negligible leakage is detected (less than 0.2% of total attached streptavidin after 2 months at 37 degrees C). However, it should be noted that not all of the four streptavidin subunits are covalently coupled to the beads. Typically, one or two of the subunits are covalently coupled. Streptavidin is like other proteins; if heated, it can denature and dissociate into subunits. If Dynabeads Streptavidin magnetic beads are, for instance, 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 unbound streptavidin molecule

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The exact number of streptavidin molecules bound per bead is not measured, but is approximately 14-16 µg streptavidin per mg Dynabeads M-280 Streptavidin magnetic beads.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The M stands for magnetic. M-280 refers to hydrophobic 2.8 micron beads, while M-270 refers to hydrophilic 2.8 micron beads. MyOne refers to 1 micron beads.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Answering this question is not straightforward. It will depend on the detection method. When using HRP (horseradish peroxidase)-based detection system or radioactivity in combination with a good antibody, very little target is required. More target is required when using an AP (alkaline phosphatase)-based detection system. When a sensitive detection system is used, detection will most likely be in the nanogram range. In some cases, pictograms of target can be detected.

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Within practical limits, the elution volume can be scaled up or down to suit your experiment. However, volumes less than 10 µL become more difficult to work with. In addition, the amount of target is important. If you have a lot of beads with a lot of bound target in a small elution volume, your elution may not be very efficient. Typically, 15-100 µL of beads may be eluted in 30 µL. For efficient recovery of the antigen and/or binding partners, the elution volume should at minimum equal the volume of the beads.

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There are several methods to quantify the amount of antibody bound to the beads. The crudest method is to measure the concentration of antibody in the coupling reaction before and after antibody attachment. Either fluorescence measurements or absorbance at 280 nm can be used. Alternatively, you could measure the amount of antibody bound to the beads by fluorescence, chemiluminescence, or radiolabeling detection methods.

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Incubation time will depend on the immunogenicity of the primary antibody and its binding affinity with the specific antigens. For a good primary antibody, 30-40 minutes incubation should work well. If you are working with a poor antibody or a very low-abundance protein, you could try to increase binding by incubating overnight. However, this also increases the chance of background protein binding.

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If the target protein has the same molecular weight as the heavy or light chain antibody, we would recommend covalently binding the antibody to the bead surface. This can be done by either crosslinking the antibody to the Dynabeads Protein A or G magnetic beads, or secondary coated beads, or by using one of the surface-activated Dynabeads magnetic beads.

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Using Dynabeads magnetic beads for protein isolation provides several advantages:

-Rapid binding kinetics: since the number of beads per volume for Dynabeads is approximately 1,000 times higher than for the same volume of a Sepharose slurry, the probability for Dynabeads magnetic beads to hit the target is far greater.

-Incubation time: due to the rapid binding kinetics, the protocol is usually very short.
-Low background: due to the rapid binding kinetics and the short incubation time, the background is also very low.
-Trapping of impurities: the beads offer no internal volume for binding or trapping of impurities.
-Low antibody consumption: this is because Dynabeads magnetic beads are nonporous, uniform superparamagnetic, monodispersed, highly crosslinked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The beads are coated with a thin layer of a highly crosslinked polystyrene shell that encases the magnetic material and prevents any leakage from the beads or trapping of ligands in the bead interior. The outer layer also provides a defined surface area for the adsorption or coupling of various molecules such as antibodies. Uniformity of bead size and shape provide consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
-Reproducibility: due to easier practical handling, such as pipetting. No centrifugation steps or preclearing are required.

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No. Not only is dithionite a reducing agent, but the strong affinity of the dithionite ion for bivalent and trivalent metal cations (M2+, M3+) allows it to enhance the solubility of iron, making it a chelating agent. As a result, the iron in the Dynabeads magnetic beads is reduced and pulled out when they are exposed to dithionite. The same is observed if Dynabeads magnetic beads are exposed to DTT and EDTA. With EDTA, we highly recommend checking the minimal amount of EDTA that your specific molecules would tolerate for binding to the Dynabeads, and if it will affect your specific application. For some applications, low concentrations of EDTA can be tolerated by Dynabeads. On the other hand, using 10 mM EDTA with heating affects the binding of biotin molecules to Dynabeads streptavidin.

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Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.

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Dynabeads magnetic beads, being magnetic in nature are really not designed to be centrifuged. That being said, the beads themselves are compact, as the pores in the polymer matrix are filled with magnetic material and coated with a final outer polymer shell that will further add to the rigidity of the beads. Hence, pressure should theoretically not be a problem for the beads themselves, but the force exerted by the beads on surrounding cells in the pellet may be detrimental to the cells.

Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Magnetic beads, unlike agarose beads, are solid and spherical, and antibody binding is limited to the surface of each bead. While magnetic beads do not have the advantage of a porous center to increase the binding capacity, they are significantly smaller than agarose beads (1 to 4 µm), which collectively gives them adequate surface area-to-volume ratios for optimum antibody binding.

High-power magnets are used to localize magnetic beads to the side of the incubation tube and out of the way to enable cell lysate aspiration without the risk of also aspirating immune complexes bound to the beads. Magnetic separation avoids centrifugation, which can break weak antibody-antigen binding and cause loss of target protein.

However, an issue with the use of magnetic beads is that bead size variations may prevent all beads from localizing to the magnet. Additionally, while immunoprecipitation using agarose beads only requires standard laboratory equipment, the use of magnetic beads for immunoprecipitation applications requires high-power magnetic equipment that can be cost-prohibitive. Read more about our Magnetic Immunoprecipitation Products (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation.html#products).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.