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View additional product information for SuperBlock™ Blocking Buffer - FAQs (37518, 37580, 37581, 37545, 37535, 37536, 37515, 37516)
15 product FAQs found
Allow any colloidal carbon, if present, to settle to the bottom of the bottle and pouring off what you need to use. We do not recommend centrifugation or filtering because these procedures may reduce the product's blocking capabilities.
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Kathon Preservative, a broad spectrum antimicrobial agent, is used at a concentration of 600 ppm.
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Its shelf life is one year at 4°C.
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Typical procedures use 1 hour at room temperature or overnight at 4°C. However, sufficient blocking in many procedures is possible in 3 x 2 minutes at room temperature.
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No, SuperBlock Blocking Buffer is not RNase-free.
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Yes, SuperBlock Blocking Buffer works with nylon and nitrocellulose membrane.
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No. It is supplied at 1X concentration and should be used “as is.“
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No. Unless it includes “T20“ in the name, for example, SuperBlock T20 (PBS) Blocking Buffer, which contains 0.05% Tween 20.
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After the plates are blocked, remove the blocking buffer and air dry the plates for several hours. Store plates with desiccant in a plastic bag at 4°C. Change desiccant after 24 hours for optimal storage. The plates should be stable for at least 12 months.
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Optimize the concentration of primary and secondary antibodies. In some cases, increasing the concentration of blocking agent (BSA or non-fat dry milk) or usiing an alternative blocking solution such as Starting Block or SuperBlock may reduce background signal. After incubation with the primary antibody, wash at least 2 times with TBST (include 0.5 M NaCl in one or more of the wash steps). Avoid Nonidet P40 or Triton X-100 in buffers because protein detection is decreased when these detergents are used.
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This information is proprietary. The protein concentration in the SuperBlock (PBS) Blocking Buffer has been optimized for direct usage without further dilution, however if desired, the blocking buffer may be diluted with phosphate-buffered saline. For best results, our recommendation is to empirically determine the optimal concentration to use.
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A blocking step should be performed to reduce fluorescence due to non-specific antibody binding. A common blocking step is the addition of a 2-5% solution of bovine serum albumin (fraction V defatted BSA). Another approach employs the addition of a 5-10% solution of serum from the species in which the secondary antibodies were raised. For example, when using goat anti-mouse IgG secondary antibodies, samples may be effectively blocked with 5-10% normal goat serum. To further reduce background fluorescence, the Image-iT FX Signal Enhancer can be included as a pre-blocking step to decrease non-specific labeling due to charge interactions between the dyes on the conjugates and the cellular constituents.
If you are using a secondary antibody make sure that the species of the antibody is not the same as the species of the sample. For example do not use an anti-mouse secondary antibody on mouse tissue.
Titrate the antibody to the lowest concentration you can use and still get adequate signal.
Try using a fluorescently tagged primary antibody because it should give reduced background but be aware this can reduce signal intensity.
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There can be many causes, including insufficient blocking, too high a concentration of the primary or secondary antibody, or degraded primary or secondary antibody. A “no-primary antibody” control can help determine if the secondary antibody is at fault. Otherwise, we recommend trying more stringent blocking or lower concentrations of primary and secondary antibodies.
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An optimal concentration may be between 1-10 µg/mL for cell and tissue labeling for microscopy, or 0.2-5 µg/mL for flow cytometry. A range of concentrations should be tested to determine what is optimal.
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For blocking cells or tissues you may use 2-5% bovine serum albumin (fraction V, defatted BSA) or 5-10% normal serum of the species matching the host species of the secondary antibody. Other options include a mixture of BSA and serum or other purified proteins. We offer a ready-made blocking solution, BlockAid Blocking Solution (Cat. No. B10710), which is not species-specific. For cell or tissue samples, avoid the use of non-fat dry milk as a blocking agent as it contains a high level of phosphoproteins, histones, and biotin.
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