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View additional product information for Dynabeads™ Goat Anti-Mouse IgG - FAQs (11033)
53 product FAQs found
请查看以下可能原因：
•溶液太粘稠。
•蛋白质间相互作用导致磁珠聚集。
尝试以下建议：
•延长分离时间（将管子留在磁力架上2-5分钟）。
•向裂解液中加入DNase I（约0.01 mg/mL）。
•将结合和/或清洗缓冲液中的Tween20浓度增加至约0.05%。
•向结合和/或清洗缓冲液中加入最多至20 mM 的β-巯基乙醇。
对于小于1 kb的生物素标记DNA，我们推荐使用Dynabeads M270链霉亲和素磁珠和MyOne C1磁珠。对于大于1kb的双链DNA分子，我们推荐Dynabeads KilobaseBINDER试剂盒。KilobaseBINDER试剂包括M-280链霉亲和素偶联的Dynabeads磁珠和一种含有专利的固定活化剂的结合液，可结合较长的生物素化DNA分子以进行分离。请点击以下链接(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)，查看关于长的生物素化DNA片段分离的更多信息。
可以，Dynabeads磁珠可用于分离单链DNA。链霉亲和素Dynabeads磁珠能够以生物素化的DNA片段为靶标，通过使双链DNA变性，从而去除非生物素化链。链霉亲和素偶联的Dynabeads磁珠不会抑制任何酶活性。因此，可以在固相上直接对磁珠结合的DNA进行下一步处理。请点击以下链接(https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html)，查看关于单链DNA捕获的更多信息。
磁化率能够衡量磁珠向磁力架迁移的速度，其大小取决于铁含量和氧化铁的特性。Dynabeads磁珠的磁化率是指质量磁化率，单位可以是cgs单位/g或m^3/kg（国际单位制）。对于亚铁磁性和铁磁性物质，质量磁化率取决于磁场强度（H），这些物质的磁化强度与H不是线性关系，而是随着场强增加而趋于饱和。因此， Dynabeads磁珠的质量磁化率是在固定条件下由标准操作程序而测定的。我们产品目录中给出的质量磁化率是国际单位制。磁化率由从高斯（cgs、emu）单位向国际单位的转换，是通过“高斯系数（emu/g或cgs/g）x 4π x 10^-3”而实现的。所得单位也被称为合理化质量磁化率，与（国际单位制）无量纲磁化率单位有所区别。通常，质量磁化率可用来衡量在非均匀磁场中影响物体的力（Fz）。测定Dynabeads磁珠的质量磁化率时，首先对样本称重，然后将样本放置于已知强度的磁场中。随后，再次称重得到样本重量（F1），并与关闭磁场时样本的重量（F0）进行对比。使用下述公式计算磁化率：K x 10^–3 = [(F1-F0) x m x 0.335 x 10^6]，K表示质量为m的样本的质量磁化率。最后，将磁化率转换为国际单位制。
有多种不同的方法可以检测配体与磁珠结合，包括光密度（OD）检测、荧光标记和放射性标记。
对于OD检测，应在配体固定到磁珠上之前检测配体的OD值，并将其与包被后上清液中剩余的配体浓度进行比较。这样可以粗略检测有多少蛋白与磁珠结合。 实验方案： 1.将分光光度计设置到正确的波长。使用偶联缓冲液作为空白组。 2.检测偶联前溶液的吸光值。根据配体的加入量，可能需要进一步稀释以读取吸光值。 3.检测偶联后溶液的吸光值。也可能需要进一步稀释以读取吸光值。 4.计算偶联效率，以“蛋白质摄取量%”表示，如下所示：[(偶联前溶液的吸光值x D) – (偶联后溶液的吸光值x D)] x 100/(偶联前溶液的吸光值 x D)，D = 稀释倍数。 对于荧光标记，我们建议对配体结合量进行反向定量，即检测偶联上清液中剩余的配体量（与原始样本对比），而不是直接检测磁珠上的配体量。将标记的配体加入到磁珠中，并检测上清液中剩余多少配体（而不是结合到磁珠上的配体）。通过与开始时加入的总配体量相比，可以计算出结合到磁珠上的配体量。由于Dynabeads磁珠具有自发荧光，因此，我们不推荐直接检测与磁珠结合的配体的荧光，而是推荐这种间接方法。标记物可以是FITC/PE等。有些研究人员也成功使用了直接检测方法（采用流式细胞仪）。 在3种方法中，放射标记的灵敏度最高，但难度最大。该方法涉及到对配体的一部分进行放射性标记。在偶联前，使用示踪剂量的放射性标记的I-125，将其以一定比例与“冷”配体混合。使用闪烁（γ）计数器对磁珠进行检测，并将磁珠的cpm值与标准品对比，得到磁珠上配体的绝对量。 实验方案： 1.取出适量磁珠，并使用1 mL结合缓冲液清洗。 2.吸取适量人IgG，置于一个单独的管子中。 3.将人IgG与I-125标记的人IgG（30,000–100,000 cpm）混合。 4.使用结合缓冲液将人IgG与I-125标记的人IgG混合物稀释至100mL。 5.室温下孵育30分钟，使用闪烁计数器检测cpm值。 6.清洗磁珠（和包被层）4次，再次检测cpm值。 使用下述方程计算结合率%：（清洗后cpm值/清洗前cpm值）x100%。Dynabeads磁珠有3种尺寸：4.5 µm （M-450）、2.8 µm （M-270/M-280）和1 µm （MyOne beads）。其中最大尺寸的磁珠非常适合细胞等较大的目标，2.8 µm磁珠推荐用于蛋白质组学和分子研究，而最小的1 µm磁珠则适用于自动化处理。
一般来说，在加入配体包被磁珠时，短时间超声是减少磁珠聚集、确保磁珠获得最佳均一性的好方法。一旦目标分子结合到磁珠，就要加倍小心了，以防结合被破坏。链霉亲和素磁珠本身能够承受超声。超声5分钟是可以的，更长时间超声的影响还未被测试。关于链霉亲和素-生物素的相互作用可否被超声破坏目前也尚无相关信息。
只有未包被的环氧基或甲苯磺酰基活化的磁珠可根据需要使用70%乙醇进行清洗除菌。包被的磁珠不可灭菌。
Dynabeads磁珠是一种大小均一、无孔、超顺磁的、单分散的、高度交联的聚苯乙烯微球，整个磁珠由均匀分散的磁性材料构成。该磁性材料由磁赤铁矿(γ-Fe2O3)和磁铁矿(Fe3O4)的混合物组成。在Dynabeads磁珠M-280和M-450中，铁(Fe)分别占磁珠重量的12%和20%。Dynabeads磁珠表面覆盖有一层薄的聚苯乙烯外壳，将磁性材料包裹在内，可防止磁珠泄漏或在内部捕获配体。此外，该外壳也可避免目标分子直接接触铁，同时为每次实验提供特定的表面来吸附或偶联各种分子。
磁珠尺寸和形状均一，确保物理和化学性质稳定一致，进而提高实验结果的质量和可重复性。
Dynabeads磁珠分为3种不同尺寸：4.5 μm （M-450磁珠），2.8 μm （M-270/M-280磁珠）和1 μm （MyOne磁珠）。
可以。请参见此海报（https://tools.thermofisher.com/content/sfs/posters/Exosome-poster-ISEV-2013-Boston.pdf）。
此外，这里还有一些相关引用：
•Blood 91:2573 (1998)
•Science 289:444 (2000)
•J Physiol 537:537 (2001)
•Mol Cell Proteomics 12:587 (2013)
•Biol Reprod 81:717 (2009)
是的，基于不同的膜表面标志物所分离外泌小体中的蛋白表达谱之间会有明显差异。这一结论由Tauro等（http://www.ncbi.nlm.nih.gov/pubmed/23230278）的研究证实，该研究团队基于EpCAM或A33这两种膜表面标志物，从人癌细胞系的条件培养基中分离出两群明显不同的外泌小体。蛋白组学研究结果显示这两群外泌小体是独特的。
我们拥有多款外泌小体分离试剂盒，包括外泌小体—人源CD63（货号10606D），外泌小体—人源CD9（货号10614D），外泌小体—人源CD81（货号10616D）和外泌小体—人源EpCAM，适用于凭借这些常用的外泌小体膜表面抗原来实现外泌小体分离操作。如果您希望使用您的自备抗体通过其他特异性膜表面标志物来分离外泌小体，您也可使用我们的Dynabeads外泌小体免疫沉淀（ProteinA，货号10610D），Dynabeads外泌小体免疫沉淀（ProteinG，货号10612D）或用于分离/检测的外泌小体—链霉亲和素产品（货号10608D）。此外，用户也可选用抗小鼠IgG的Dynabeads磁珠（货号11031或11033）和识别特定膜表面标志物的小鼠单抗来实现外泌小体分离操作。
一般通过流式细胞仪（使用CD9、CD63、TSG101和Alix等膜表面标志物）来鉴定外泌小体，通过EM来研究其形态和尺寸，或凭借LC-MS/MS来实现更为详细的蛋白分析。
这些标志物要基于外泌小体的细胞来源进行选择。最常用于外泌小体分离和鉴定的膜表面标志物为CD9、CD63、CD81或TSG101。下表列举了一些最近用于鉴定或分离外泌小体的参考文献和膜表面标志物：
膜表面标志物
参考文献
Alix, CD63, EpiCam, HSP70, TSG101
Mol Cell Proteomics 12:587 (2013)
CD9, CD63
Hum Mol Genet 21:R125 (2012)
CD63, MHC II
J Biol Chem 278:52347 (2003)
CD9, CD81, Lamp1, TSG101
Cancer Res 67:7458 (2007)
CD63
Nature Cell Biol 9:654 (2007)
Alix, CD37, CD53, CD63, CD81, CD82, TSG101
J Cell Biol 200:373 (2013)
CD59, CD63, CD133, TSG101
FASEB J 23:1858 (2009)
除沉淀法之外，外泌小体还可通过超速离心或密度梯度分离法实现分离。用户也可使用靶向外泌小体标志物（例如人源的CD9、CD63、CD81、EpCAM）的Dynabeads磁珠或二抗包被的Dynabeads磁珠（使用靶向其他外泌小体膜表面标志物的不同抗体），凭借磁性方法来分离外泌小体。
外泌小体被报道具有多种不同的功能，如抗原呈递、凋亡、血管发生、炎症和凝血作用，这些作用是通过蛋白/脂类交换或信号途径的激活来实现的。外泌小体提供了一种胞间遗传物质交换的全新机制，并能够介导细胞间的相互通讯。外泌小体也能够转运和传播传染性物质，如朊蛋白和逆转录病毒。
外泌小体是微小的卵形或杯形膜结构，大小在30-150 nm，其中包含有mRNA，microRNA，蛋白和脂类。外泌小体可由正常，异常或肿瘤细胞释放进入血、尿、唾液和乳汁等体液中。外泌小体源于内吞型细胞器，并作为与质膜融合的多泡体（MVB）而从细胞释放出来(J Cell Biol 200:373 (2013)).
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.
The secondary-coated Dynabeads magnetic beads can be coupled to a primary antibody by a direct or an indirect approach.
Direct approach: The Dynabeads magnetic beads are first coupled with your primary antibody and then used for isolating your target cell type.
Indirect approach: The cells are first incubated with your primary antibody(ies). The Dynabeads magnetic beads are then added to the antibody-coated target cells.
Secondary-coated Dynabeads magnetic beads can be used in several cell isolation approaches:
Cell depletion--using an antibody to target the unwanted cell type and a secondary-coated Dynabeads magnetic beads product.
Negative cell isolation--using a cocktail of antibodies to target all unwanted cell types and a secondary-coated Dynabeads magnetic beads product (using the indirect approach).
Positive cell isolation without detachment--using an antibody to target the wanted cell type and a secondary-coated Dynabeads magnetic beads product.
Positive cell isolation with detachment--using an antibody to target the wanted cell type and a CELLection Dynabeads magnetic beads product.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
You may use one of our secondary-coated, surface-activated, or streptavidin-coated Dynabeads magnetic beads, and coat it with a primary antibody to target your cell type.
The Dynabeads magnetic beads product you choose will depend on the primary antibody available for cell targeting and the downstream application for the isolated cells:
-For primary antibodies made in mouse, use the CELLection magnetic beads Pan Mouse IgG Kit, Dynabeads magnetic beads Goat Anti-Mouse IgG, Dynabeads magnetic beads Pan Mouse IgG, Dynabeads magnetic beads Rat Anti-Mouse IgM, Dynabeads magnetic beads Rat Anti-Mouse IgM, or Dynabeads magnetic beads Sheep-Anti Mouse IgG
-For primary antibodies made in rat, use the Dynabeads magnetic beads Sheep Anti-Rat IgG
-For primary antibodies made in rabbit, use the Dynabeads magnetic beads M-280 Sheep Anti-Rabbit IgG
-For primary antibodies made in any species, use the CELLection magnetic beads Biotin Binder Kit, Dynabeads magnetic beads Biotin Binder, Dynabeads magnetic beads FlowComp Flexi, Dynabeads magnetic beads M-450 Epoxy, or Dynabeads magnetic beads M-450 Tosylactivated
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Yes, a cocktail of primary antibodies can be added to a cell suspension in order to pull out several target cell populations with one secondary-coated Dynabeads magnetic beads product.
The Dynabeads magnetic beads Pan Mouse IgG (110.41; 110.42) works very well with a cocktail of mouse IgGs for the simultaneous capture of multiple cell types. it is recommended that you use an indirect technique with antibody cocktails (add all Ab to cells, wash off excess Ab, then add beads to capture Ab-coated cells).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The indirect technique is chosen when the antigen targeted by the primary antibody is expressed in low density on the target cell surface. This is due to the fact that free antibodies will find their target antigen more easily than antibodies linked to the Dynabeads magnetic beads. Also when using the indirect technique, an excess of free antibody can be added to the system, allowing ample opportunity for monoclonals to find the target antigen. Finally, an indirect technique can be useful when a cocktail of monoclonal antibodies is used to deplete unwanted cells during negative isolation of a cell type. This is because antibodies against all unwanted cell types can be added at once to the starting cell population, provided the antibodies are from one species. The antibody-coated cells can then be targeted with secondary-coated Dynabeads magnetic beads.
The direct technique is chosen when a limiting amount of monoclonal antibody is needed for targeting the cells of interest during positive isolation or depletion (e.g., when the target antigen is present at high density). It can also help when the possibility of interaction from the secondary antibody needs to be avoided, or if a stock preparation of primary coated Dynabeads magnetic beads is desired. Additionally the direct technique can be used when you do not want to cover all antigen sites with antibody (e.g., when you want to analyze the isolated cells by flow cytometry).
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When a secondary-coated Dynabeads magnetic beads product is used for negative isolation or depletion of cells you can choose between the following techniques.
Direct technique
-Add primary IgG antibody to the secondary-coated Dynabeads magnetic beads with specificity to the species of your primary IgG antibody
-Add the resulting primary coated Dynabeads magnetic beads to cells for capture and separation
Benefits:
-You can make up a stock of beads
-Use less primary antibody
-Save time, fewer steps and is easier to optimize than the indirect procedure
-Lower background, additional steps increase the chance of nonspecific signal
-Lower cost, since direct procedures require much lower amounts of tag specific antibodies than indirect procedures
OR
Indirect technique
-Add primary IgG antibody to the cells
-Wash cells to remove excess, unbound antibody
-Add secondary-coated Dynabeads magnetic beads with specificity to the species of your primary IgG antibody to capture primary antibody coated cells
Benefits:
-You can use a cocktail of antibodies to coat different cell types i.e., a negative isolation approach
-Can be used when target antigen expression is low for more efficient binding of antibody to cells.
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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.
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Yes. The antibodies are covalently bound and should be very stable in your applications.
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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 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.
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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.
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.
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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.
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.
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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).
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Yes. See this poster (https://tools.thermofisher.com/content/sfs/posters/Exosome-poster-ISEV-2013-Boston.pdf).
In addition, here are some citations:
- Blood 91:2573 (1998)
- Science 289:444 (2000)
- J Physiol 537:537 (2001)
- Mol Cell Proteomics 12:587 (2013)
- Biol Reprod 81:717 (2009)
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Yes, exosomes isolated with different surface markers can be distinctive in their protein profile. This has been demonstrated by Tauro et al. (http://www.ncbi.nlm.nih.gov/pubmed/23230278), who isolated two distinctive populations of exosomes based on surface markers EpCam or A33 from conditioned cell culture medium from a human carcinoma cell line. This proteomics study indicated that these two populations of exosomes are unique.
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We have exosome isolation kits for Exosome-Human CD63 (Cat. No. 10606D), Exosome-Human CD9 (Cat. No. 10614D), Exosome-Human CD81 (Cat. No. 10616D), and Exosome-Human EpCAM intended for isolating exosomes with these commonly used exosome surface antigens. If you are interested to isolating exosomes with other specific surface markers using your own antibody, you can use our Dynabeads exosome immunoprecipitation (Protein A, Cat. No. 10610D), Dynabeads exosome immunoprecipitation (Protein G, Cat. No. 10612D), or Exosome-Streptavidin for isolation/detection (Cat. No. 10608D). In addition, anti-mouse IgG Dynabeads magnetic beads (Cat Nos. 11031 or 11033) also can be employed in exosome isolation using mouse monoclonal antibodies against selected surface markers.
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Exosomes are usually characterized by flow cytometry (using surface markers such as CD9, CD63, TSG101, and Alix), by EM to study morphology and size, or by detailed protein analysis by LC-MS/MS.
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It depends on the cell source from which the exosomes were derived. The most commonly used surface markers for isolating and characterizing exosomes are CD9, CD63, CD81, or TSG101. Here are some of the recent references and surface markers for identifying or isolating exosomes:
Alix, CD63, EpiCam, HSP70, TSG101 Mol Cell Proteomics 12:587 (2013)
CD9, CD63 Hum Mol Genet 21:R125 (2012)
CD63, MHC IIJ Biol Chem 278:52347 (2003)
CD9, CD81, Lamp1, TSG101 Cancer Res 67:7458 (2007)
CD63 Nature Cell Biol 9:654 (2007)
Alix, CD37, CD53, CD63, CD81, CD82, TSG101J Cell Biol 200:373 (2013)
CD59, CD63, CD133, TSG101 FASEB J 23:1858 (2009)
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Exosomes can be isolated by ultracentrifugation or density gradient separation in addition to a precipitation approach. Exosomes can also be isolated by a magnetic approach using Dynabeads magnetic beads targeting exosome markers such as Human CD9, CD63, CD81, EpCAM or secondary antibody-coated Dynabeads magnetic beads using different antibodies against other exosomal surface markers.
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A range of different functions have been reported such as antigen presentation, apoptosis, angiogenesis, inflammation, and coagulation by protein/lipid exchange or activation of a signaling pathway. Exosomes provide a novel vehicle for genetic exchange between cells and mediate cell to cell communication. Exosomes also transport and propagate of infectious cargoes such as prion and retrovirus.
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Exosomes are small, membrane-bound ovoid to cup shaped particles around 30-150 nm in size containing mRNA, microRNA, proteins, and lipids. Exosomes are released by normal, abnormal, or neoplastic cells into body fluid such as blood, urine, saliva, and breast milk. Exosomes originate from the endocytic compartment and are released from cells as multivesicular bodies (MVB) fused with plasma membrane (J Cell Biol 200:373 (2013)).
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