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弯曲条带以及我们该如何进行处理?

弯曲条带以及我们该如何进行处理?

If you’ve worked with SDS-PAGE and Western blotting for a while you’ll undoubtedly have seen bendy or fuzzy bands, so unlike those perfect razor-sharp rectangles shown on your antibody datasheet. While unattractive in their own right these bendy bands also make determining the molecular weight difficult, can interfere with band densitometry analysis, especially when using automated programs or when working with proteins that produce tight duplexes.

如果你曾经做过SDS-PAGE和Western blotting实验,你肯定会看到弯曲的或模糊的条带,不同于你的抗体数据表上显示的那些完美的条带形状。虽然这些弯曲的条带本身并不吸引人,但它们会影响到分子量,这可能会影响到条带密度测量的分析,尤其是在使用自动化程序或分析分子量相近的蛋白

Fear not though, as there are typically five extremely simple steps you can take which will cure the vast majority of band bending, and make your blots presentable again.

不要担心,通常您可以采取五个非常简单的步骤,使您的完美印迹再次呈现。

Clean your kit

清洁实验器材

You’ve spent weeks growing your rare cells, treated them with your incredibly expensive novel compound, carefully harvested and lysed your sample and then cast your gel in between two dirty smeared plates. Ensuring that your plates and combs are spotlessly clean before casting is a great step in ensuring your perfect gels and perfect blots.

你花了几个星期的时间来培养你的稀有细胞,用昂贵的新化合物来处理它们,小心地收集和溶解你的样本,然后却把你的凝胶放在两个脏的玻璃板之间。所以要确保你的玻璃板和梳子在铸造之前是干净的,这是确保你铸造完美凝胶非常重要的一步骤。

Shaken and stirred

摇动和搅拌

But then left to rest. A major issue with gel casting is improper mixing of reagents, in particular the acrylamide. In order to ensure a smooth separation of proteins across your resolving gel you want to ensure a constant acrylamide percentage throughout. But equally you want to get your gels cast and on with your experiment as quickly as possible. But here, as with many things in life more haste equals less

speed. A thorough mixing of gel reagents, followed by the equally important degassing step will give you pristine gels first time, every time.

凝胶铸造的一个主要问题是不适当的混合试剂,特别是丙烯酰胺。为了确保在凝胶中均匀地分离蛋白质,需要确保一个恒定的丙烯酰胺百分比。但同样的,你也要尽可能快地将配好的胶用于实验。一次彻底的混合凝胶试剂,接着是同样重要的脱气步骤,每次都能给你纯洁的凝胶。

Fresh is best

保持试剂新鲜

In the same way, it’s worth keeping on top of your buffers and ensuring they’re fresh and importantly have the correct pH. We often have breaks in our Western blotting, and whilst it’s tempting to reach for that 6 month old bottle of running buffer, which has been sitting in the sun, it probably makes more sense to throw it out and start again. At the very least checking the pH of your stacking and resolving gel reagents is a must as this plays a fundamental role in how your proteins migrate through the gel.

相同的方式,缓冲buffer确保它们是新鲜的,重要的是调整到正确的PH值。我们经常发现我们的western blotting是重复不出来,使用将近6个月不新鲜的缓冲buffer,并将buffer一直放在阳光下,可能把buffer扔掉重新配置更有意义。至少要检查一下buffer的pH值是必须的,因为它在您的蛋白质迁移中通过凝胶发挥重要作用。

Slow and steady

低电压慢跑

As above, we know you want to get your exciting results ASAP, but running your gels with a high voltage might leave you having to cast and re-run your samples again. Forcing your samples through the stack too quickly can mean they hit the resolving gel spread out, rather than in a nice tight line. The same is true in the resolving gel, by running your gels at too high a voltage the buffers and your gels become hot, and this is a major contributor to warping and bending in your gel.

如上所述,我们知道你希望尽快获得令人兴奋的结果,以高电压运行您的凝胶可能会导致您不得不再配胶并重新运行样品。通过在高电压下运行你的凝胶,缓冲液和凝胶会变热,这是您的凝胶翘曲和弯曲的主要原因。

More isn’t always better

上样要适中

Less about bending and more about blotting, this step is vital in ensuring you get the most accurate results possible from your blotting. While it’s tempting to load the maximum concentration of protein in the maximal volume, sometimes it’s worth reducing the load to ensure you get a nice sharp band rather than an unintelligible blot. With new antibodies, and even new antibody batches, it’s always worth running a test with a standard curve of total protein. This simple step can save you sample, and time as it makes your analysis quicker and easier.

减少关于条带弯曲和更多关于印迹,这一步对于确保您从印迹获得最准确的结果至关重要。尽管在最大体积中加载最大浓度蛋白质是有诱惑力的,但有时候减少上样量,可以确保您获得一个不错的条带而不是无法辨认的印迹。使用新的抗体,甚至是新的抗体批次,需要运用总蛋白质标准曲线进行测试。这个简单的步骤可以节省您的时间和样品,使您的分析更快捷。

在这五个步骤之后,你可以让你的条带变得锐利,让它们成为你的实验室的骄傲。

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