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SPL Frequently asked questions (FAQs)

SPL Software-993x1024

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What is the difference between a (fluorescent) stain and a Smart Label?

A stain is a dye, which binds non-covalently to proteins. Staining requires time for staining and destaining and is very limited in terms of reproducibility. Smart Labels are fluorescent dyes, which bind covalently to proteins. Smart labeled proteins can be visualized directly after electrophoresis – no staining or destaining steps are required. Smart Labels are highly sensitive with a wide dynamic range.

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Is the Smart Labeling exclusive for certain proteins or will it favor certain proteins?

Smart Labels bind to all proteins in a non-saturated way to allow for precise quantification. The labeling chemistry and ratio of labels to proteins are well established (see more than 1,000 DIGE and Refraction-2D publications) and guarantees an even labeling of all proteins within one sample.

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What may inhibit the labeling process?

Due to special SPL buffers, the Smart Label reaction is very robust. Smart Labeling is compatible with all commonly used buffer systems. However, high concentrations of reducing agents (> 15 mM DTT or > 10 % beta-mercaptoethanol) and amines > 400 mM may interfere with labeling reaction. The SPL Smartalyzer (SMA) is added to the labeling reaction as a control. Its unique design monitors labeling efficiency and if necessary compensates for unequal labeling.

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For sample Smart Labeling, what is the maximum and minimum volume of my sample? The maximum volume is 10 µl per Smart Label reaction (containing up to 20 µg of protein). The minimum volume is limited only to the volume you can handle.

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If I have to dilute my protein sample before Smart Labeling, can I use my own buffer? Yes please.

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What do I have to do, if I want to label more than 20 µg of protein per sample?Multiply the Smart Labeling reaction (e.g. 21 – 40 µg of protein sample – use two Smart Label reaction assays per sample.

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Why is the Smartalyzer (SMA) bi-fluorescent?

 

The SMA itself is mono-fluorescent (e.g. SMA basic blue). SMA is added to the protein sample before Smart Labeling. SMA is labeled together with the protein sample (e.g. with Smart Label Red). So SMA becomes bi-fluorescent (e.g. basic blue and label red). SMA basic allows for normalization of gel loading and SMA label allows for normalization of labeling.

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Which size of the standard SMA should I use, 12.5 kDa or 80 kDa?

The size of the SMA should differ from the size of your target protein and highly abundant proteins within your sample.

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How do I avoid that the SMA size S (12.5 kDa) runs out of the gel?

Stop electrophoresis before the dye front runs out of the gel. Cut the dye front manually before fluorescence imaging (dye front may interfere with Imaging).

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Does the Smart Label interfere with subsequent Mass Spectrometry?

No. Smart Labels do not effect enzymatic digestions nor sequence coverage.

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Does the Smart Label interfere with antibody recognition?

No. Only one amino acid of a minimal amount of protein is labeled.

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Can I stain Smart labeled gels and then cut out bands?

Yes. Smart Labels are compatible with all commonly visual protein stains (e.g. Coomassie® or silver nitrate) and subsequent protein identification by mass spectrometry.

 

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