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View additional product information for Dynabeads™ Streptavidin Magnetic Beads - FAQs (65604D, 65801D, 65605D, 65002, 65001, 65602, 65305, 65601, 11205D, 11206D, 65306, 65606D, 65607D, 60101, 60210)
45 product FAQs found
Please review the following possibilities for why your Dynabeads magnetic beads are not pelleting:
- The solution is too viscous.
- The beads have formed aggregates because of protein-protein interaction.
Try these suggestions:
- Increase separation time (leave tub on magnet for 2-5 minutes)
- Add DNase I to the lysate (~0.01 mg/mL)
- Increase the Tween 20 concentration to ~0.05% of the binding and/or washing buffer.
- Add up to 20 mM beta-merecaptoethanol to the binding and/or wash buffers.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
For biotin-labeled DNA that is less than 1 kb, we recommend you use Dynabeads M270 Streptavidin (Cat. No. 65305) and MyOne C1 magnetic beads (Cat. No. 65001). We recommend our Dynabeads KilobaseBINDER Kit (Cat. No. 60101), which is designed to immobilize long (>1 kb) double-stranded DNA molecules. The KilobaseBINDER reagent consists of M-280 Streptavidin-coupled Dynabeads magnetic beads along with a patented immobilization activator in the binding solution to bind to long, biotinylated DNA molecules for isolation. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html) for more information in regards to long biotinylated DNA fragment isolation.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Yes, Dynabeads magnetic beads can be used to isolate single-stranded DNA. Streptavidin Dynabeads magnetic beads can be used to target biotinylated DNA fragments, followed by denaturation of the double-stranded DNA and removal of the non-biotinylated strand. The streptavidin-coupled Dynabeads magnetic beads will not inhibit any enzymatic activity. This enables further handling and manipulation of the bead-bound DNA directly on the solid phase. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html) for more information in regards to single-stranded DNA capture.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Magnetic susceptibility is a measure of how quickly the beads will migrate to the magnet. This will depend on the iron content and the character of the iron oxide. The magnetic susceptibility given for the Dynabeads magnetic beads is the mass susceptibility, given either as cgs units/g or m^3/kg (the latter being an SI unit). For ferri- and ferromagnetic substances, the magnetic mass susceptibility is dependent upon the magnetic field strength (H), as the magnetization of such substances is not a linear function of H but approaches a saturation value with increasing field. For that reason, the magnetic mass susceptibility of the Dynabeads magnetic beads is determined by a standardized procedure under fixed conditions. The magnetic mass susceptibility given in our catalog is thus the SI unit. Conversion from Gaussian (cgs, emu) units into SI units for magnetic mass susceptibility is achieved by multiplying the Gaussian factor (emu/g or cgs/g) by 4 pi x 10^-3. The resulting unit is also called the rationalized magnetic mass susceptibility, which should be distinguished from the (SI) dimensionless magnetic susceptibility unit. In general, magnetic mass susceptibility is a measure of the force (Fz) influencing an object positioned in a nonhomogenous magnetic field. The magnetic mass susceptibility of the Dynabeads magnetic beads is measured by weighing a sample, and then subjecting the sample to a magnetic field of known strength. The weight (F1) is then measured, and compared to the weight of the sample when the magnetic field is turned off (F0). The susceptibility is then calculated as K x 10^-3 = [(F1-F0) x m x 0.335 x 10^6], where K is the mass susceptibility of the sample of mass m. The susceptibility is then converted to SI units.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
There are different methods to check binding of ligands to the beads, including optical density (OD) measurement, fluorescent labeling, and radioactive labeling.
For OD measurement, you would measure the OD of the ligand before immobilization to the beads and compare it with the ligand concentration that is left in the supernatant after coating. This gives a crude measurement of how much protein has bound to the beads.
Protocol:
1.Set spectrophotometer to the right wavelength. As a blank, use the Coupling Buffer.
2.Measure the absorbance of the Pre-Coupling Solution. A further dilution may be necessary to read the absorbance, depending upon the amount of ligand added.
3.Measure the absorbance of the Post-Coupling Solution. A dilution may be necessary to read the absorbance.
4.Calculate the coupling efficiency, expressed as the % protein uptake, as follows. [(Pre-Coupling Solution x D) - (Post-Coupling Solution x D)] x 100/(Pre-Coupling Solution x D) where D = dilution factor.
For fluorescent labeling, we suggest negatively quantifying the amount of ligand bound by measuring ligand remaining in the coupling supernatant (compared to the original sample), rather than directly measuring the ligands on the beads. Add labeled ligand to the beads, and measure how much ligand is left in the supernatant (not bound to the beads). By comparing this with the total amount added in the first place, you can then calculate how much of the ligand that has been bound to the beads. Keep in mind that the Dynabeads magnetic beads are also autofluorescent, which is why direct measuring of fluorescence of the bead-bound ligands is not recommended, but rather this indirect approach. The label could be, for example, FITC/PE. Some researchers perform a direct approach with success (using a flow cytometer).
Radioactive labeling is the most sensitive method of the three, but it is also the most difficult one. It involves radioactively labeling a portion of the ligand. We use radiolabeled I-125 in tracer amounts and mix it with "cold" ligands in a known ratio before coupling. The absolute quantities for the ligand on the beads should be obtained by measuring the beads in a scintillation (gamma) counter and comparing the cpm with a standard.
Protocol:
1.Take out an appropriate amount of beads and wash the beads in 1 mL of binding buffer.
2.Pipette out desired amount of human IgG in a separate tube.
3.Mix the human IgG with I-125-labeled human IgG (30,000 - 100,000 cpm).
4.Dilute the mixture of human IgG and I-125-labeled human IgG to 100 mL in binding buffer.
5.Incubate for 30 minutes at room temperature and measure the cpm in a scintillation counter.
6.Wash the beads (with coating) four times, and measure cpm again.
The % binding is calculated by using the equation : (cpm after washing/cpm before washing)x100%.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads come in three sizes: 4.5 µm (M-450), 2.8 µm (M-270/M-280), and 1 µm (MyOne beads). The largest of the Dynabeads magnetic beads is ideal for big targets like cells. The 2.8 µm beads are recommended for proteomics and molecular applications. The smallest of the beads, 1 µm, are ideal for automated handling.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification 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.
In general, short sonication is a good way to reduce aggregation of the beads and ensure optimal homogenous conditions at the time of ligand addition when coating the beads. When target is bound to the beads, more care is needed, as the binding might break. The streptavidin beads themselves should tolerate sonication. We have not tested sonication for long periods, but 5 minutes is fine. We do not have information about the streptavidin-biotin interaction being broken by such treatment.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
If desired, the uncoated epoxy or tosylactivated beads can be sterilized by washing with 70% ethanol. Coated beads cannot be sterilized.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly cross-linked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic material within the Dynabeads magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4). The iron content (Fe) of the beads is 12% by weight in Dynabeads magnetic beads M-280 and 20% by weight in Dynabeads magnetic beads M-450. The Dynabeads magnetic beads are coated with a thin polystyrene shell which encases the magnetic material, and prevents any leakage from the beads or trapping of ligands in the bead interior. The shell also protects the target from exposure to iron while providing a defined surface area for the adsorption or coupling of various molecules.
Uniformity of bead size and shape provides consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
The Dynabeads magnetic beads are available in three different sizes: 4.5 µm (M-450 beads), 2.8 µm (M-270/M-280 beads) and 1 µm (MyOne 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.
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 (Cat. No. 65001) 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.
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.
The Dynabeads KilobaseBINDER Kit (Cat. No. 60101) is designed for the binding of large (greater than 2 kb) DNA or RNA fragments. The kit contains Dynabeads M-280 Streptavidin magnetic beads plus a unique and patented binding solution. With this binding solution, it is possible to immobilize 70 pmol of a 4 kb DNA fragment and 80 pmol of a 10 kb fragment per mg Dynabeads magnetic beads. This enables various applications to be conducted, including pure template isolation for gene expression, protein-DNA interaction studies, and mRNA processing studies.
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.
For isolating large fragments of dsDNA, the Dynabeads kilobaseBINDER Kit should have much higher binding capacity (200-600 µg DNA per mg Dynabeads magnetic beads) than the Dynabeads MyOne Streptavidin C1 magnetic beads (~20 µg per mg Dynabeads magnetic beads). The larger the dsDNA, the more advantage the Dynabeads kilobaseBINDER Kit format would have (this is in large part due to the buffer system).
Both of these Dynabeads products have excellent signal:noise ratio, but the 1 µm beads (Dynabeads MyOne Streptavidin C1 magnetic beads) sediment slower so they work better in automated 96-well format. Conversely, the 2.8 µm beads (Dynabeads kilobaseBINDER magnetic beads) are faster to bind to the magnet and easier to manually handle.
Basically, for large total amounts of dsDNA oligos or smaller fragments, for samples with high chaotropic salt concentration, and if you are interested in avoiding BSA, we recommend the Dynabeads MyOne Streptavidin C1 magnetic beads (Cat. No. 65001). For large ds DNA fragments (>4 kb), we recommend the Dynabeads kilobaseBINDER Kit (Cat. No. 60101).
Superparamagnetic means that the Dynabeads magnetic beads exhibit magnetic properties when placed within a magnetic field, but have no residual magnetism when removed from the magnetic field.
This means that your targeted cells, proteins, or nucleic acids are only subjected to magnetic forces during the time the beads are on the magnet. The beads do not aggregate, but remain evenly dispersed in suspension.
Find additional tips, troubleshooting help, and resources within our Dynabeads Cell Isolation and Expansion Support Center.
Yes. The antibodies are covalently bound and should be very stable in your applications.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Depending on the antibody coated on the Dynabeads magnetic beads, the shelf life can vary from 24-36 months.
Some kits may have 18 months shelf life depending on other components supplied in the kit. The kits are guaranteed for 6 months from when you receive them.
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 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.
Currently, this buffer is only sold as part of the kit and is not available separately
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.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
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.
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.
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.
Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
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.
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.
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.
Find additional tips, troubleshooting help, and resources within ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
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.
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.
Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.