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The first step in this kit is to bind all endogenous biotinylated entities with an unlabeled streptavidin. The free D-biotin is added to bind to all biotin binding sites on the added streptavidin. The free biotin will be washed away before any other labels are added and all biotin binding sites will be occupied on the unlabeled streptavidin.

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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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Here are some suggestions: Use the Qdot Incubation Buffer (Cat. No. Q20001MP). The included buffer is formulated specifically for improved signal-to-background ratios in most immunolabeling applications using the Qdot streptavidin conjugates. Alternate buffers may result in more variable staining and, in particular, may increase background staining. However, some specific applications may require other buffer conditions. Please see the protocol "Double-labeling Using Qdot Streptavidin conjugates."
Determine if the sample has a high level of endogenous biotin. Block the sample using an avidin-biotin pre-blocking step.
If you have used the Qdot Incubation Buffer and still get high nonspecific background, then it may be necessary to check other steps of your procedure. Blocking the sample with BSA or normal animal serum will generally decrease nonspecific binding of both antibodies and Qdot streptavidin conjugates. It is a good practice to dilute your primary and secondary antibodies in the blocking buffer. Some tissues such as spleen and kidney sections may contain endogenous biotin, which may contribute to non-specific signal. Endogenous biotin can be blocked with an avidin/biotin blocking kit (Cat. No. E21390).
Grainy staining or clumps of fluorescent material appear in the background.
Occasionally the BSA within the Qdot Incubation Buffer shows slight aggregation over time. It is necessary to remove this aggregate prior to labeling the sample with the Qdot streptavidin conjugate. Spin down the incubation mixture before addition to the sample. This can be accomplished by spinning the samples in a benchtop centrifuge (Eppendorf 5415) at 5,000 x g for 2 minutes. The material can also be passed over a 0.2 µm spin filter unit before you add it to the sample for staining to remove microscopic precipitates. If you are using a buffer that is different than the Qdot Incubation Buffer, this behavior can often be attributed to higher levels of NaCl or other salts in the incubation buffer, and may not be easily fixed with filtration. In this case, reduce the overall salt concentration.
Optimize concentration of biotinylated secondary antibodies.
Optimizing specific signal can often be achieved by adjusting the level of biotinylated antibody used instaining. High levels of biotinylated antibody are necessary to obtain specific labeling, but overly high levels will contribute to nonspecific binding of the antibody to the sample. Nonspecifically bound biotinylated antibody will bind to the Qdot streptavidin conjugate, resulting in higher staining of the background.
Optimize concentration of Qdot streptavidin conjugate.
Just as titration of primary and secondary antibodies is necessary to achieve optimal specific signal in immunolabeling applications, the level of the final probe should be optimized for each conjugate. In general, concentrations at or slightly below saturation should have the optimal signal-to-background ratio, while concentrations substantially higher than saturation will compromise the assay with higher background levels.

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The number of molecules conjugated to one Qdot nanocrystal is based on the ratio of quantum dot:molecule used in the conjugation, the number of available binding sites on the Qdot nanocrystal, and the size of both the Qdot nanocrystal and the molecule of interest. In general, there are 2-3 antibodies, 4-5 biotin molecules, and 6-8 streptavidin molecules per Qdot nanocrystal.

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We have four types of streptavidin-coated Dynabeads magnetic beads. They differ in size, binding capacity, and surface charge. These four beads are based upon two different chemistries, tosyl chemistry and carboxylic acid chemistry. Streptavidin is covalently coupled to these functional groups in order for biotin to bind. These beads cannot be used for further coupling to remaining tosyl or carboxylic acid groups on the beads. We have two medium-sized 2.8 micron beads. One is based upon a tosyl-activated bead, meaning that it is a hydrophobic bead (Dynabeads M-280 Streptavidin Beads, Cat. No. 112-05D), and the other is based upon a carboxylic acid bead, meaning that it is hydrophilic (Dynabeads M-270 Streptavidin Beads, Cat. No. 65305). We also have a 1 micron alternative based upon tosyl-activated beads (Dynabeads MyOne Streptavidin T1 Beads, Cat. No. 65601) and another based upon carboxylic acid (Dynabeads MyOne Streptavidin C Beads, Cat. No. 65001), meaning that they will differ in hydrophobicity and binding capacity.

Dynabeads M-280 Streptavidin magnetic beads works in applications such as immunoassays, purification of DNA/RNA binding proteins, protein purification, and cell isolation.

Dynabeads M-270 Streptavidin magnetic beads works in the same applications, such as nucleic acid assays, but also in protein isolation.

Dynabeads M-280 Streptavidin magnetic beads: binding capacity of 650-900 pmol/mg beads of free biotin, binding capacity of 5-10 µg/mg beads of biotinylated IgG

Dynabeads MyOne Streptavidin T1 magnetic beads:

binding capacity of more than 1,300 pmol/mg beads of free biotin, binding capacity of 10-25 µg/mg beads of biotinylated IgG

Dynabeads M-270 Streptavidin magnetic beads: binding capacity of 650-1,350 pmol/mg beads of free biotin, binding capacity of 5-10 µg/mg beads of biotinylated IgG

Dynabeads MyOne Streptavidin C1 magnetic beads: binding capacity of more than 2,500 pmol/mg beads of free biotin, binding capacity of 15-20 µg/mg beads of biotinylated IgG

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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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No, both Biotin Binder and streptavidin Dynabeads magnetic beads are coated with wild type streptavidin, which binds DSB-X biotin too tightly to be efficiently released by free normal biotin.

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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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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.

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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.

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Dynabeads FlowComp kits are based on modified biotin-streptavidin isolation and release technology. The capture antibodies are labeled with DSB-X biotin, while the Dynabeads magnetic beads in the Dynabeads FlowComp kits are coated with modified streptavidin. The DSB-X biotin on the target antibody has a lower affinity to the streptavidin than normal biotin. Thus, following the positive isolation, the cells are released by adding the release buffer containing excess of normal biotin that out-competes the binding between the modified streptavidin on the Dynabeads magnetic beads and the DSB-X biotin-labeled target antibody. As a result, the isolated cells are bead-free but not antibody-free (the DSB-X biotinylated target antibody is still attached to the cell surface).

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Steric hindrance can interfere with the binding. As with avidin, the double ring structure of biotin must fit 9 angstroms into the streptavidin molecule for binding to occur. Competitive inhibition by endogenous biotin may also interfere with binding of your biotinylated ligand.

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- 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.

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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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Non-specific binding can often be relieved by a blocking solution, but microspheres seem to require a stronger blocking solution than those most commonly commercially available. Hence, we've developed the BlockAid Blocking Solution (Cat. No. B10710). This reagent is a protein-based blocking solution designed for use with FluoSpheres microspheres and TransFluoSpheres microspheres conjugated to biotin, streptavidin, NeutrAvidin biotin-binding protein, or other proteins. The BlockAid Blocking Solution has proven useful for reducing the nonspecific binding of protein-coated or other macromolecule-coated microspheres in a wide variety of flow cytometry, microscopy, and microarray applications.

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