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查看更多产品信息 SuperBlock™ Blocking Buffer - FAQs (37518, 37580, 37581, 37545, 37535, 37536, 37515, 37516)
28 个常见问题解答
将所有存在的胶态碳沉至瓶底，然后将您需要使用的溶液倒出。我们不建议离心或过滤，因为这些操作可能会使产品的封闭能力降低。
卡松防腐剂，一种广谱抗微生物剂，浓度为600 ppm。
在4℃下的有效期为1年。
常规程序是在室温孵育1小时或在4℃孵育过夜。但是，在很多程序中，在室温下孵育3 x 2分钟足以实现有效封闭。
不是，SuperBlock封闭缓冲液不是RNase-free的。
可以， SuperBlock封闭缓冲液能和尼龙膜和硝酸纤维素膜一起工作。
不需要。该产品以1X浓度供应，应“按原液”使用。
不含。除非是名字中含有“T20”的封闭缓冲液，如SuperBlock T20（PBS）封闭缓冲液，含有0.05% Tween 20。
微孔板经过封闭处理后，去除封闭缓冲液并风干数小时。将微孔板与干燥剂一起装在塑料袋中，保存于4℃。为实现最佳的保存，应在24小时后更换干燥剂。微孔板可稳定保存至少12个月。
•需要进行封闭操作以减少由非特异性抗体结合产生的背景荧光。常用的封闭步骤是加入2-5％牛血清白蛋白（fraction V defatted BSA）溶液。另一种方法是采用5-10％二抗种属来源的血清溶液。例如，使用山羊抗小鼠IgG二抗时，样品可以被5-10％正常山羊血清有效封闭。为了进一步减少背景荧光，可以使用Image-iT FX 信号增强剂作为预封闭步骤，减少由偶联物上的染料和细胞组分之间电荷的相互作用引起的非特异性标记。
•如果您使用二抗，确保抗体的种属与样品不同。例如不要对小鼠组织使用抗小鼠二抗。
•滴定测定抗体浓度，使用能获得充足的信号的最低浓度。
•试试荧光标记的一抗，因为它应会降低背景干扰，但这样做也会降低信号强度。
原因可能很多，包括封闭不足、一抗或二抗浓度过高或一抗或二抗降解。通过“无一抗”对照加以确定是否是二抗的问题。否则，我们建议你们尝试更严格的封闭或更低浓度的一抗或二抗。
进行细胞和组织标记显微检测的最佳浓度为1-10 µg/mL，进行流式细胞术的最佳浓度为0.2-5 µg/mL。最佳浓度需通过试验确定。
如要封闭细胞或组织，可以使用2-5％的牛血清白蛋白（第五组分，脱脂BSA）或是与二抗宿主种属相匹配的5-10％正常血清。其他选择还包括BSA和血清或其他纯化蛋白的混合物。我们提供一种没有种属特异性的即用型封闭液，即BlockAid封闭液（货号B10710）。对于细胞或组织样品，要避免使用脱脂奶粉作为封闭剂，因为它含有大量的磷蛋白、组蛋白和生物素。
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.