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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)
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我们不推荐这样做，否则链霉亲和素变性时会变为疏水性并发生聚集。
高背景可能是由磁珠表面的BSA非特异性结合或者链霉亲和素的非特异性结合引起的。升高pH或盐浓度，都有助于减少非特异结合。建议改用Dynabeads M-270链霉亲和素磁珠，这些磁珠无BSA包被，且由于其是基于羧基活化的而具有亲水性。
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。
一般来说，我们不建议冷冻Dynabeads磁珠，因为冻融过程会使磁珠表面形成裂缝，导致从磁珠中释放铁，污染样本。反复冷冻/融化过程会加剧该效应，必须避免。对于表面包被了抗体的Dynabeads磁珠，尤其需要注意，否则会损坏这种磁珠表面抗体的性能。我们推荐将Dynabeads磁珠在2-8℃直立放置保存，确保磁珠浸没于缓冲液中（干燥会降低性能）。始终切记，在使用前一定要完全重悬磁珠并妥善清洗。
选择哪种产品取决于样本的性质、使用的缓冲液和溶液以及下游应用。通常，所有Dynabeads链霉亲和素磁珠都可用于生物素化配体相关的应用；然而，在特殊应用中，有些磁珠可能因其特性而比其他磁珠的效果更好。Dynabeads M-280链霉亲和素磁珠和Dynabeads MyOne链霉亲和素T1磁珠常用于蛋白质和核酸相关应用。Dynabeads M-270链霉亲和素磁珠和MyOne链霉亲和素C1磁珠更适用于核酸诊断，特别是用于含有高浓度高离液盐的样本、涉及小的生物素化抗原的免疫分析以及与BSA不相容的应用，因为这些磁珠没有包被BSA。Dynabeads MyOne链霉亲和素磁珠具有更强的结合能力和更慢的沉降速度，使其适用于自动化应用以及需要分离大量生物素化化合物或其特异性靶标的情况。请点击这里(https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html?icid=fr-strep-1)查看选择指南。
您可通过检测上清液中未结合的核酸量，从而得到与Dynabeads链霉亲和素磁珠结合的核酸量。通过OD检测或凝胶电泳可获知核酸浓度。也可以对核酸进行放射性标记以直接在磁珠上检测核酸浓度，或通过荧光标记核酸而在上清液中检测浓度。
我们的Dynabeads 链霉亲和素磁珠可直接用于PCR或real-time PCR。然而，你必须根据经验来优化每一反应体积中所用的磁珠的数量。
尚未检测过每个磁珠结合的链霉亲和素分子精确数量，但每毫克Dynabeads M-280链霉亲和素磁珠大约结合14-16 µg链霉亲和素。
所有生物素试剂都应包含一个间隔臂，至少为一个6-碳接头，以减少空间位阻。这是因为生物素的双环能够进入到链霉亲和素的生物素结合槽深处。一个6碳臂是在生物素和序列第一个碱基之间能够减少空间位阻所需的最短长度。该距离越长，空间位阻越小。6-碳接头是大多数公司采用的标准接头尺寸，应该能够满足大部分应用需求。我们推荐在探针的5’端进行特定的生物素化。
在直接捕获中，生物素化探针/配体先与Dynabeads磁珠偶联，随后再加入样品。在间接捕获中，先将生物素化探针/配体加入到样品中，随后再加入Dynabeads磁珠。直接捕获中预先偶联的配体使得Dynabeads磁珠能够重复使用，而间接捕获在目标分子浓度较低、特异性亲和力较弱或结合动力学速度慢的情况下更有益。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/nucleic-acid-capture-assays.html)，查看捕获图解。
1 mg Dynabeads M-280链霉亲和素磁珠通常可结合650–900 pmol游离生物素、200 pmol生物素化多肽、最多10 μg生物素化抗体、10 μg生物素化双链DNA或200 pmol生物素化单链寡核苷酸。 1 mg Dynabeads M-270链霉亲和素磁珠通常可结合950 pmol以上游离生物素、200 pmol生物素化多肽、最多10 μg生物素化抗体、10 μg生物素化双链DNA或200 pmol生物素化单链寡核苷酸。 1 mg Dynabeads MyOne链霉亲和素C1磁珠通常可结合2,500 pmol以上游离生物素、400 pmol生物素化多肽、最多20 μg生物素化抗体、20 μg生物素化双链DNA或500 pmol生物素化单链寡核苷酸。 1 mg Dynabeads MyOne链霉亲和素T1磁珠通常可结合1,100–1,700 pmol游离生物素、400 pmol生物素化多肽、最多20 μg生物素化抗体、20 μg生物素化双链DNA或400 pmol生物素化单链寡核苷酸。
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。
这取决于涂层或生物素化分子的特性。我们建议您通过测试来确定针对特定实验的最佳条件。
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）。
可通过碱处理或高温处理分离DNA双链。采取碱处理时，可使用0.1 M NaOH洗脱非生物素化链。通常情况下，该处理方法不会对磁珠产生任何影响。
•在NaOH处理前，在2X结合和洗涤缓冲液（10 mM Tris-HCl，pH 7.5，1 mM EDTA，2 M NaCl）中清洗Dynabeads/DNA复合物1次，除去上清液。高盐浓度将有助于减少电荷，从而使非特异性结合最小化。
•向Dynabeads/DNA复合物中加入新鲜配制（很重要）的0.1 M NaOH溶液，室温下旋转孵育2-3分钟（最多5分钟）。除去含有非生物素化链的上清液。
•再用0.1 M NaOH溶液清洗Dynabeads/DNA复合物1次，除去上清液。首次洗脱时，即可洗脱下大部分DNA。
除了碱处理，也可进行加热处理，在水中95°C加热5分钟即可。
为防止再退火，碱处理或高温处理后必须快速从磁珠上分离DNA，最好在冰上操作。加热会在一定程度（在水中约为7%）上引起生物素化DNA从链霉亲和素上解离。因此，我们通常更推荐采用碱处理法。
打破生物素-链霉亲和素的结合需要苛刻的条件。为了从Dynabeads链霉亲和素磁珠中分离生物素化DNA，我们建议在10mm EDTA, 95%甲酰胺，pH 8.2中，在65摄氏度下加热5分钟或在90摄氏度下加热2分钟。或者，把Dynabeads-DNA复合物在0.1%的SDS中煮沸5分钟。
Dynabeads链霉亲和素磁珠不是以无RNase溶液的形式提供的。处理RNA时，应使用DEPC处理的0.1 M NaOH/0.05 M NaCl溶液清洗磁珠2次，每次1-3分钟。DEPC毒性很强，但能去除RNase。清洗后，可使用DEPC处理的0.1 M NaCl溶液重悬磁珠。
“DEPC处理”是指：加入0.1% DEPC，混合，室温下孵育1小时。随后，将DEPC处理的溶液进行高压灭菌，以破坏DEPC。
We do not recommend this as streptavidin becomes hydrophobic and aggregates during denaturation.
The background might be caused by nonspecific binding to the BSA on the bead surface. Alternatively, high background might be caused by nonspecific binding to streptavidin. Increasing either the pH or the salt concentration might help reduce the binding. Dynabeads M-270 Streptavidin magnetic beads might be a better alternative; these beads are not coated with BSA and are hydrophilic, as they are based upon carboxylic acid chemistry.
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.
Yes, you can still use them. To test stability at low temperature in-house, the Dynabeads M-280 Streptavidin beads were placed at -20 degrees C for 24 hrs and then transferred to room temperature (15-25 degrees C) for 72 hrs. This freeze/thaw cycle was repeated 4 times and the beads were stored at 2-8 degrees C for 60 months. The biotin binding capacity was then tested and was seen to be within our specifications.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Which product to choose depends on the properties of your sample, the buffers and solutions applied, as well as the downstream application. In general, all Dynabeads Streptavidin beads can be used in applications involving biotinylated ligands; however, some beads may perform better than others in particular applications due to their characteristics. Dynabeads M-280 Streptavidin beads and Dynabeads MyOne Streptavidin T1 beads are commonly used for protein and nucleic acid applications. Dynabeads M-270 Streptavidin beads and MyOne Streptavidin C1 beads are preferred for nucleic acid diagnostics, specifically with samples that have a high concentration of chaotropic salts, immunoassays involving small biotinylated antigens, and in applications that are not compatible with BSA, as these beads are not blocked with BSA. Dynabeads MyOne Streptavidin beads offer increased binding capacity and slower sedimentation rate, making them ideal for automated applications and when larger amounts of a biotinylated compound or its specific target need to be isolated. Please see the selection guide here ( https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html?icid=fr-strep-1).
You can assay the supernatant for unbound nucleic acid to determine the amount of nucleic acid bound to the Dynabeads Streptavidin beads. The concentration of nucleic acids can be checked by measuring the OD or by running them on a gel. Alternatively, the nucleic acids can be labeled radioactively and the concentration measured directly on the beads, or fluorescently and measured in the supernatant.
Our Dynabeads Streptavidin magnetic beads can be used directly in PCR or real-time PCR. However, you would have to empirically optimize the amount of beads to be used per volume of reaction.
The exact number of streptavidin molecules bound per bead is not measured, but is approximately 14-16 µg streptavidin per milligram Dynabeads M-280 Streptavidin magnetic beads.
All biotin reagents should contain a spacer arm, at least a 6-carbon linker, to reduce steric hindrance. This is because the bicyclic ring of biotin goes deep into the biotin binding cleft in streptavidin. A 6-carbon arm is the minimum length between biotin and the first base of sequence that is required to reduce the steric hindrance effect. The longer this distance is, the less the steric hindrance. A 6-carbon linker is standard linker size from most companies and should be enough for most applications. We recommend specific biotinylation at the 5'-end of the probe.
In direct capture, the biotinylated probe/ligand is first coupled to the Dynabeads magnetic beads followed by addition of your sample. In indirect capture, the biotinylated probe/ligand is first added to the sample followed by addition of your Dynabeads magnetic beads. Precoupled ligand for direct capture allows you to reuse the Dynabeads magnetic beads, while an indirect approach can be beneficial when the concentration of your target is low, specific affinity is weak, or the binding kinetics is slow. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/nucleic-acid-capture-assays.html) for a diagram of the capture.
- One milligram of Dynabeads M-280 Streptavidin magnetic beads typically binds 650-900 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 µg of biotinylated antibody, 10 µg of biotinylated double-stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.
- One milligram of Dynabeads M-270 Streptavidin magnetic beads typically binds more than 950 pmol of free biotin, 200 pmol of biotinylated peptide, up to 10 µg of biotinylated antibody, 10 µg of biotinylated double- stranded DNA, or 200 pmol of biotinylated single-stranded oligonucleotides.
- One milligram of Dynabeads MyOne Streptavidin C1 magnetic beads typically binds more than 2,500 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 µg of biotinylated antibody, 20 µg of biotinylated double-stranded DNA, or 500 pmol of biotinylated single-stranded oligonucleotides.
-One milligram of Dynabeads MyOne Streptavidin T1 magnetic beads typically binds 1,100-1,700 pmol free biotin, 400 pmol of biotinylated peptides, up to 20 µg of biotinylated antibody, 20 µg of biotinylated double- stranded DNA, or 400 pmol of biotinylated single-stranded oligonucleotides.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
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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.
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If desired, the uncoated epoxy or tosylactivated beads can be sterilized by washing with 70% ethanol. Coated beads cannot be sterilized.
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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.
The background might be caused by nonspecific binding to the BSA on the bead surface. Alternatively, background might be caused by nonspecific binding to streptavidin. Increasing either the pH or salt concentration might help reduce the binding. Dynabeads M-270 Streptavidin magnetic beads(Cat. No. 65305) might be a better alternative; these beads are not coated with BSA and have a hydrophilic surface, as they are based upon carboxylic acid chemistry.
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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.
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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.
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The streptavidin molecule is covalently attached to the bead's surface. However, not all of the four streptavidin subunits are covalently coupled to the beads, typically only one or two are covalently coupled. Streptavidin is like other proteins; if heated it can denature and dissociate into subunits. If streptavidin-coupled Dynabeads are boiled, some of the streptavidin subunits may be released (as monomers or aggregates) from the beads. The covalently bound streptavidin subunits will not be affected by such treatment. When streptavidin is bound to biotin, the streptavidin-biotin complex is more stable than the streptavidin itself. Under normal, recommended conditions, only negligible leakage of streptavidin from the beads is detected (less than 0.2% of total attached streptavidin after 2 months at 37 degrees C).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Streptavidin is a protein made up of four identical subunits, each containing a high affinity binding site for biotin (KD = 10-15 M). Streptavidin has the same biotin binding properties as avidin, but less nonspecific binding is observed. After immobilization on the beads, there are 2-3 binding sites free for interaction with biotin.
Find additional tips, troubleshooting help, and resources within ourDynabeads Nucleic Acid Purification Support Center as well as ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
The Dynabeads KilobaseBINDER Buffer is a viscous solution and therefore the beads will behave differently in this buffer than in other buffers. Be patient while resuspending the beads in the buffer and try to avoid pumping air into the tube. Flicking the tube containing the beads and buffer must be done very carefully.
If still not completely dissolved, leave it on a roller at 4 degrees C.
Dynabeads M-280 Streptavidin magnetic beads are supplied at 10 mg (approx. 6.7 x 10e8) beads per mL, dissolved in phosphate buffered saline (PBS), pH 7.4, containing 0.1% BSA and 0.02% NaN3 as a preservative.
Dynabeads M-270 Streptavidin magnetic beads are supplied at both 10 mg (approx. 6.7 x 10e8) beads per mL and at 50 mg (approx. 3.2 x 10e9) beads per mL. Both products are dissolved in PBS, pH 7.4, containing 0.02% NaN3 as a preservative.
Dynabeads MyOne Streptavidin C1 magnetic beads are supplied at 10 mg (approx. 7-12 x 10e9) beads per mL, dissolved in PBS, pH 7.4, containing 0.01% Tween 20 detergent and 0.09% NaN3 as a preservative.
Dynabeads MyOne Streptavidin T1 magnetic beads are supplied at 10 mg (approx. 7-12 x 10e9) beads per mL, dissolved in PBS, pH 7.4, containing 0.01% Tween 20 detergent and 0.02% NaN3 as a preservative.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Dynabeads M-270 Streptavidin magnetic beads and Dynabeads MyOne C1 magnetic beads have a negatively charged surface. The surface charge of the beads may in some samples cause the beads to float or become sticky or aggregate. The stickiness may be due to electrostatic interactions between the beads or between the beads and the tube wall. Usually we recommend washing the beads in a nonionic detergent like Tween 20 detergent before doing the experiment. The problem is usually reduced or eliminated by simply adding Tween 20 detergent to a final concentration of up to 0.1% to the beads, followed by resuspension and washing in buffer without the detergent. An incubation in the Tween 20 solution may be needed, e.g. 5-10 min at room temperature on a roller. In addition, we recommend using siliconized tubes. This treatment will most likely reduce the electrostatic potential of the beads.
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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.
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The isoelectric point for Dynabeads M-270 Streptavidin magnetic beads is pH 4.5 and for Dynabeads M-280 Streptavidin magnetic beads it is pH 5.0.
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Storage should be at 2 to 8 degrees C. Freezing or drying of the Dynabeads magnetic beads is not recommended. Provided the Dynabeads magnetic beads are stored correctly, quality is guaranteed until the expiry date stated on the label.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The binding capacity of streptavidin-coupled Dynabeads magnetic beads is fragment length-dependent. Reduced binding capacity for large DNA fragments may be due to steric hindrance. For large DNA fragments (greater than 2 kb in size), we recommend using Dynabeads kilobaseBINDER Kit.
-Salt concentration affects the binding efficiency of biotinylated nucleic acids to the Streptavidin-coupled Dynabeads magnetic beads. Optimal binding conditions for biotinylated DNA fragments (up to 1 kb) are achieved at 1 M NaCl (final concentration), 25 degrees C and 15 min incubation time. Longer DNA fragments should be immobilized overnight. Biotinylated antibodies should be immobilized in PBS buffer pH 7.4, supplemented with 0.1% BSA.
-Ensure that your sample does not contain an excess of free biotin, as the free biotin will bind Streptavidin-coupled Dynabeads magnetic beads much more rapidly than larger biotinylated molecules. Biotinylated oligonucleotides should be recovered by reverse phase HPLC or FPLC to remove free biotin from the sample.
-We also recommend a titration to optimize the quantity of beads used for each individual application, since both the size of the specific molecule to be immobilized and the biotinylation procedures affect the binding capacity of the beads.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
A low pH elution buffer, such as 0.1 M glycine-HCl, pH 2.5-3.0, would be a good solution for eluting the antigen without eluting the antibody off the beads.
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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.
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When exposed to a sample consisting of different types of molecules, any solid phase matrix can interact with these molecules due to hydrophobicity, charge or other types of interactions. It is not surprising that you get non-specific binding to the beads. This method is actually used for pre-clearing of sample and is not considered a good negative control. When pre-blocked and coated with a specific molecule, beads show a lot less non-specific binding than when they are not coated. As a negative control, you could try beads that are coated with an irrelevant molecule.
Find additional tips, troubleshooting help, and resources within ourProtein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
This buffer is intended for immobilizing oligo on to the beads in the absence of protein or when the direct method is applied. For the indirect method, when the oligo is first mixed with the protein sample and Dynabeads M-280 Streptavidin are added to capture DNA-protein complex, a buffer with salt concentration as low as 150 mM should be applied. The most common methods are to use either a high salt buffer or boil the Dynabeads with DNA-protein complex in SDS sample buffer for 3-4 mins. With a high salt buffer, a salt concentration higher than 1 M is normally applied to break DNA-protein interaction. The exact amount of salt required depends on the affinity of the protein for oligonucleotides and should be determined for each application.
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There are two methods to dissociate the non-biotinylated DNA from the biotinylated DNA strand. The following protocols are based on using 20 µL of Dynabeads Streptavidin, but are scalable. Both methods may release very small amounts of complementary biotinylated strand from streptavidin. If it is critical that no biotinylated strand is released, either adopt a different biotin modification using dual biotin (two biotin groups in sequence) or covalently bind DNA to e.g., Dynabeads M-270 Carboxylic Acid.
Using heat:
- Wash the DNA coated Dynabeads in 50 µL 1 x SSC.*
- Resuspend the beads in another 50 µL of 1 x SSC Incubate at 95 degrees C for 5 mins.
- Quickly put the tube in magnet stand for 1-2 mins and transfer the supernatant to a new tube.
- The supernatant contains your non-biotinylated DNA strand.
Using NaOH:
- Wash the DNA coated Dynabeads in 50 µL 1 x SSC.*
- Resuspend the beads in 20 µl of freshly prepared 0.15 M NaOH.
- Incubate at room temperature for 10 mins. Put the tube in magnet stand for 1-2 mins and transfer the supernatant to a new tube.
- The supernatant contains your non-biotinylated DNA strand. Neutralize the probe by adding 2.2 µL 10 x TE, pH 7.5 and 1.3 µL 1.25 M acetic acid.
Wash the Dynabeads coated with biotinylated strand once with 50 µL 0.1M NaOH, once with 50 µL of B&W buffer and once with 50 µL TE buffer.
*1 x SSC (0.15 M NaCl, 0.015 M sodium citrate. Dissolve the reagents in 800 mL water. Adjust pH to 7.0 with NaOH. Adjust the volume to 1 liter with water).
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Assay the supernatant for unbound molecules. This will determine the amount of molecule bound to the Dynabeads. For nucleic acids, the concentration can be checked by OD readings, or by running a gel. For proteins, the concentration in the supernatant can be determined by a spectrometer using a protein assay like BCA. Alternatively, you can label the molecule with radioactivity or fluorescence and measure the concentration of molecule directly on the beads (former) or in the supernatant (latter).
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Dynabeads M-280 Streptavidin has been extensively used for this application so this is the product we recommend.
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PBS is the recommended immobilization buffer for biotinylated proteins or other molecules. For immobilization of biotinylated nucleic acids, we recommend the following Binding and Wash (B&W) buffer: 10.0 mM Tris-HCl (pH 7.5) 1.0 mM EDTA 2.0 M NaCl.
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Streptavidin is a protein composed of four identical subunits, each containing a high affinity binding site for biotin (K-D = 10 -15 M) . Streptavidin has the same biotin binding properties as avidin, but it has a low isoelectric point (pI=5) and no carbohydrate groups, resulting in low non-specific binding.
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This will depend on the properties of your sample, the buffers and solutions used and your downstream application. For an overview, see Streptavidin-Coupled Dynabeads (https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html). In general, they can all be used, but some may perform better than others in particular applications due to their characteristics.
- Dynabeads M-280 Streptavidin (Cat. No. 11205D) and Dynabeads MyOne Streptavidin T1 (Cat. No. 65601) are used for both protein and nucleic acids applications.
- Dynabeads M-270 Streptavidin (Cat. No. 65305) and Dynabeads MyOne Streptavidin C1 (Cat. No. 65001) are preferred for molecular diagnostics and for handling samples with high concentration of chaotropic salt, as well as in immunoassays involving small biotinylated antigens and in applications that are not compatible with BSA as these beads are not blocked with BSA.
- The Dynabeads Streptavidin Trial Kit (Cat. No. 65801D) allows you to try 1 mL of all four different streptavidin-coated Dynabeads products making it more convenient to test out which of the beads perform best in a specific application. The kit contains 1 mL each of Dynabeads M-280 Streptavidin, Dynabeads M-270 Streptavidin, Dynabeads MyOne Streptavidin C1, and Dynabeads MyOne Streptavidin T1.
- The Dynabeads MyOne Streptavidin C1/T1 offer an increased binding capacity and slower sedimentation rate, making them ideal for automated applications and/or for when larger amount of biotinylated molecules or their specific target need to be isolated.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Use mild elution conditions, e.g., a buffer with high salt or low pH. Heating the beads at 95 degrees C for 5 mins in SDS sample buffer will elute the antibody as well.
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The streptavidin-biotin interaction is the strongest known non-covalent biological interaction between a protein and a ligand. Binding of biotin and streptavidin is very rapid and, once formed, the complex is unaffected by wide extremes of pH, temperature, organic solvents, and other denaturing agents. Normally, very harsh methods are required to dissociate the biotin from streptavidin, which will be irreversibly denatured by the procedure. Biotin-streptavidin interactions are more easily reversible when biotin derivatives with lower binding affinity are used along with chemically modified streptavidins with lower biotin binding affinity. When modified biotins and streptavidins are used, gentle methods for inducing reversible binding are available
To dissociate biotinylated proteins from streptavidin, boil the beads in 0.1% SDS or SDS-PAGE buffer for 3 mins or incubate them in 8 M guanidinium hydrochloride, pH 1.5.
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We do not recommend this because streptavidin becomes hydrophobic and aggregates during heat-induced denaturation.
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The product to choose depends on the properties of your sample, the buffers and solutions you will use, as well as your downstream application. In general, all the Dynabeads Streptavidin beads can be used in applications involving biotinylated ligands. However, some beads may perform better than the others in particular applications due to their characteristics. Please refer to the Dynabeads Streptavidin Selection Guide (https://www.thermofisher.com/us/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads.html#selection) for detailed information.
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The streptavidin molecule is covalently attached to the surface of the beads; under normal, recommended conditions, negligible leakage is detected (less than 0.2% of total attached streptavidin after 2 months at 37 degrees C). However, it should be noted that not all of the four streptavidin subunits are covalently coupled to the beads. Typically, one or two of the subunits are covalently coupled. Streptavidin is like other proteins; if heated, it can denature and dissociate into subunits. If Dynabeads Streptavidin magnetic beads are, for instance, boiled, some of the streptavidin subunits may be released (as monomers or aggregates) from the beads. The covalently bound streptavidin subunits will not be affected by such treatment. When streptavidin is bound to biotin, the streptavidin-biotin complex is more stable than the unbound streptavidin molecule
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The exact number of streptavidin molecules bound per bead is not measured, but is approximately 14-16 µg streptavidin per mg Dynabeads M-280 Streptavidin magnetic beads.
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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.
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Using Dynabeads magnetic beads for protein isolation provides several advantages:
-Rapid binding kinetics: since the number of beads per volume for Dynabeads is approximately 1,000 times higher than for the same volume of a Sepharose slurry, the probability for Dynabeads magnetic beads to hit the target is far greater.
-Incubation time: due to the rapid binding kinetics, the protocol is usually very short.
-Low background: due to the rapid binding kinetics and the short incubation time, the background is also very low.
-Trapping of impurities: the beads offer no internal volume for binding or trapping of impurities.
-Low antibody consumption: this is because Dynabeads magnetic beads are nonporous, uniform superparamagnetic, monodispersed, highly crosslinked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The beads are coated with a thin layer of a highly crosslinked polystyrene shell that encases the magnetic material and prevents any leakage from the beads or trapping of ligands in the bead interior. The outer layer also provides a defined surface area for the adsorption or coupling of various molecules such as antibodies. Uniformity of bead size and shape provide consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
-Reproducibility: due to easier practical handling, such as pipetting. No centrifugation steps or preclearing are required.
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No. Not only is dithionite a reducing agent, but the strong affinity of the dithionite ion for bivalent and trivalent metal cations (M2+, M3+) allows it to enhance the solubility of iron, making it a chelating agent. As a result, the iron in the Dynabeads magnetic beads is reduced and pulled out when they are exposed to dithionite. The same is observed if Dynabeads magnetic beads are exposed to DTT and EDTA. With EDTA, we highly recommend checking the minimal amount of EDTA that your specific molecules would tolerate for binding to the Dynabeads, and if it will affect your specific application. For some applications, low concentrations of EDTA can be tolerated by Dynabeads. On the other hand, using 10 mM EDTA with heating affects the binding of biotin molecules to Dynabeads streptavidin.
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Yes, they are compatible with 6-8 M Urea when used during post-coupling steps.
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Dynabeads magnetic beads, being magnetic in nature are really not designed to be centrifuged. That being said, the beads themselves are compact, as the pores in the polymer matrix are filled with magnetic material and coated with a final outer polymer shell that will further add to the rigidity of the beads. Hence, pressure should theoretically not be a problem for the beads themselves, but the force exerted by the beads on surrounding cells in the pellet may be detrimental to the cells.
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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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