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View additional product information for Dynabeads™ His-Tag Isolation and Pulldown - FAQs (10104D, 10103D)
31 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.
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.
Using an EDTA solution (dissolved in water) should work, but the elution efficiency may be low due to interactions between the beads and target. You may use detergent or salt to reduce this effect, or use low pH (or low pH in combination with denaturing conditions) to increase elution efficiency.
Alternatively, EDTA may be dissolved in 10 - 100 mM Binding and Wash buffer (B&W buffer). Since the B&W buffer contains Tween detergent, proteins eluted using this solution should be gel purified if downstream mass spectrometry is to be performed.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
DTT, DTE, EDTA, and EGTA are not compatible with Dynabeads His-Tag Isolation & Pulldown magnetic beads. You should make sure that these are not present. In addition, we have some evidence that beta-mercaptoethanol and MgCl2 may have an impact on the cobalt as well.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
BSA would only prevent non-specific adsorption to the surface. Also, if the BSA binds to the chelator, it will block His-tag binding. Instead, we recommend that you use 0.01% Tween 20 detergent instead.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
If the protein of interest is expressed cytoplasmically, you may lyse the cells according to the following protocol keeping everything on ice with pre-chilled buffer:
Scrape/trypsinize the cells into single-cell suspension. Wash cells once in 3 mL PBS.
Remove all PBS and add a volume of the following buffer: 50 mM Na-phosphate, 300 mM NaCl, pH 8.0, 0.01 % Tween 20 detergent, 0.5% to 1% Triton-X 100 detergent + protease inhibitors of your choice (200 µL of this buffer for a T25 flask or 300-500 µL for T75 flask).
Mix by tapping and store on ice for 10-15 min until all cells are lysed.
Centrifuge at 2,500 x g at 4 degrees C for 10 min to remove cell debris.
Transfer the cleared supernatant to a fresh tube and follow the standard His-tagged protein purification protocol in the package insert. From this point forward, all washing steps should be performed with the standard Binding/Wash buffer, unless you are working with a membrane protein. In this case, add 0.5-1% Triton-X 100 detergent to the below buffer (the exact Triton-X 100 detergent concentration should be determined by titration).
50 mM Na-phosphate, 300 mM NaCl pH 8.0, 0.01 % Tween 20 detergent.
Since it is a eukaryotic lysate, titration with imidazole and NaCl in the Binding/Wash buffer may be necessary. Concentrations of 5-20 mM imidazole and/or up to 500 mM NaCl can be tried.
Note that purifying His-tagged proteins from mammalian total cell lysates generally gives higher backgrounds than bacteria lysates because mammalian cells have a higher level of endogenous proteins with polyhistidine motifs. Purity can be improved by having the protein secreted into the cell culture medium. If you are using this type of expression system, you can add the Dynabeads magnetic beads directly to the cell culture medium after buffering (ensure the cell media is pH ~8). Then wash the beads as specified in the package insert.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Isolation of His-tagged proteins from mammalian cells is problematic due to the endogenous expression of proteins containing polyhistidine tags in mammalian cells. We have not developed a protocol specifically for mammalian systems. To maximize recovery of the recombinant protein (over the endogenous proteins), the expression level of the recombinant protein should be sufficiently high. Given the rapid binding kinetics of the Dynabeads magnetic beads, it is very important to keep the incubation time short (5 to 10 mins) as increasing the incubation time will increase the background. For some proteins, binding efficiency might be affected by which end of the protein the His-tag is on (C or N terminal). In order to reduce background, imidazole (typically 5 mM, but up to a maximum of 20 mM) may be added in the washing solution. Also increasing the linker between the tag and the protein could be considered.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Prior to mass spectrometry, we recommend that you elute the sample from the Dynabeads magnetic beads and then further purify it by gel electrophoresis and band excision. The extra gel purification step is necessary to remove the Tween® 20; detergent, which can interfere with accurate mass spectrometry.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
For isolation of His-tagged proteins, we offer the Dynabeads His-Tag Isolation & Pulldown beads (Cat. Nos. 10103D, 2 mL (40 mg beads/mL) and 10104D, 10 mL (40 mg beads/mL)).
Binding capacity and purity:
Capacity: approximately 40 µg His-tagged GFP per milligram of beads
Purity: close to 100% (assessed by bioanalyzer)
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This principle is based on the reversible interaction between various amino acid side chains and immobilized metal ions. Depending on the immobilized metal ion, different side chains can be involved in the adsorption process. Most notably, histidine, cysteine, and tryptophan side chains have been implicated in protein binding to immobilized transition metal ions and zinc. Histidines exhibit highly selective binding to certain metals and have great utility in immobilized metal affinity chromatography (IMAC). At physiological pH, histidine binds by sharing electron density of the imidazole nitrogen with the electron-deficient orbitals of transition metals. Although only three histidines may bind transition metals under certain conditions, six histidines reliably bind transition metals in the presence of strong denaturants such as guanidinium. Such protein tags are commonly referred to as 6xHis tags. For isolation of His-tagged proteins, we offer the Dynabeads His-Tag Isolation & Pulldown beads (Cat. No. 10103D, 2 mL (40 mg beads/mL) and Cat. No. 10104D, 10 mL (40 mg beads/mL)). These beads have a cobalt-based Immobilized Metal Affinity Chromatography (IMAC) surface chemistry. The technology is comprised of a tetradentate metal chelator in which four of cobalt's six coordination sites are occupied. The imidazole rings of histidine residues present in a poly histidine peptide chain are able to occupy the two remaining coordination sites, resulting in protein binding.
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.
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.