Blot TIN in 600

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Aug 6th, 2022
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blot TIN in 600 by following these steps:

  1. Register your DocHub account or sign in if you already have one.
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  3. Select the ability to blot TIN in 600 from the menu bar and use it to the document.
  4. Go through your document again to ensure that you haven’t missed any mistakes or typos. When you finish, hit DONE.
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How to blot TIN in 600

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Western blots assess how much of a particular protein is present in a sample. The thicker or darker the band, the more of the protein is present in the sample.
Loading too much protein leads to signal saturation in western blots, yet too little produces weak signals. This protocol describes an assay development experiment to determine the appropriate protein load for target and control detection prior to performing the actual western blot experiment.
The ideal total protein loaded will vary between samples and target proteins but is typically between 1050 g. If the protein is in low abundance in the sample you will need to load a greater amount of total protein.
To obtain linear signals with the majority of western blots, we recommend loading smaller amounts of protein sample between 1 and 10 g per well. To avoid under- or overloading samples, determine the protein concentration of each sample prior to electrophoresis with a compatible protein assay.
Ideally, it is best to load 2 g per well of a purified protein or 20 g of a complex mixture like whole cell lysates if you are doing Coomassie stain only. Protein loading can be adjusted ingly for more sensitive stains like silver and fluorescent staining or when doing WB where you can do lower amounts.
We recommend sample loads between 1 and 10 g of protein per well. Optimize primary and secondary antibody dilutions. In most cases, this means reducing the total amount of antibody used (higher dilutions). This will limit the amount of HRP in the system to prevent saturation of signal.
Protein samples for western blotting can be soluble protein fluids, cell/tissue lysates or immunoprecipitated proteins. The protein loading differs from different samples, basically, the recommended protein loading of purified protein is no more than 100 ng, and the loading of cell/tissue lysate could be 10-40 g.
Western Blot Maximum Loading Volume For example, a 10-well gel (1.0mm thickness) loading volume ranges from 1 to 44 l. If pre-cast gels are used, please refer to the manufacturers instructions as the maximum well volume differs between brands.

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