How can I obtain the date of manufacture for SeeBlue Pre-Stained Standard from the lot number?
The lot number will be in the yr-mm-day format. For example, Lot No. 10820 was made August 20, 2001. One note: this convention is not necessarily adopted on the labels for all Thermo Fisher Scientific products.
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How should the SeeBlue Pre-Stained protein standard appear after silver or Coomassie staining?
In the SeeBlue Pre-Stained standard, since the dye is covalently bound to the proteins, staining will not remove the color. After Coomassie staining, the bands may appear a bit fuzzier, an observation that is consistent for all prestained standards, regardless of manufacturer. When silver stained, most of the proteins will retain some of their original blue color, with a faint brownish tint.
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What is the expected Western transfer efficiency of the SeeBlue Prestained Standard?
With the recommended load of 5 µL per lane on a mini gel and using optimal Western transfer procedures, the expected transfer efficiency is 75% below the 100 kDa range and 50% above the 100 kDa range. This is because prestained standards generally do not stick to the membranes as well as regular proteins. This is largely due to the attached dye molecules themselves, which can carry a charge or block the hydrophobic interactions that drive binding of protein to membrane.
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Can I store the SeeBlue and SeeBlue Plus2 Pre-stained standards in the freezer to increase their shelf-life?
The recommended storage condition for SeeBlue and SeeBlue Plus2 Pre-stained standards is at 4°C. Freeze-thaw cycles, which could result from the standards being shuttled between the bench and the freezer for each use, could degrade the standards over time. If the standards are to be frozen, aliquot them into single use volumes to avoid repeated freeze-thaw.
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Why are the molecular weight values for the proteins in your prestained standards such as SeeBlue and SeeBlue Plus2 different in different gel types?
The molecular weight values that are stated in conjunction with our standards are given as "apparent" molecular weights. The differences between the apparent molecular weights and the published molecular weights are a result of the covalent attachment of dye to the proteins used in the marker. The bound dye molecules can carry a charge. Of course, this charge changes the ability of the SDS to bind to the protein in addition to contributing directly to the protein's charge. The result is a protein with an altered charge and consequent change in mobility within the gel.
This explains why the proteins in prestained markers run differently from their unstained counterparts. However, it fails to fully explain why there is further difference observed between the same marker in differing gel types (Tris-Glycine vs NuPAGE gels, for example). The reason for this disparity is the different pHs of the gel types. At higher pH values (Tris-Glycine gels), charges are more likely to be protonated. Meanwhile, at the lower, more neutral pH of a NuPAGE gel, the charges are more skewed towards deprotonation, giving the same stained proteins a more negative charge overall. In an SDS PAGE system, more negative charge means more mobility. This is why the same prestained protein will be "larger" on a Tris-Glycine gel than on a NuPAGE gel. In a NuPAGE gel, the lower (approximately neutral) pH causes more overall negative charge, making the apparent molecular weight much lower. This effect generally increases in magnitude with the size of the protein and is greatest with myosin due to the increased number of dye binding sites.
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