Nanjing Xinfan Bio: Protein gel dyes are selected as you like - Huaqiang Electronic Network

Like DNA gel electrophoresis, protein gel electrophoresis is also a common task in the laboratory. Whether it is Western blot or mass spectrometry, protein electrophoresis is inseparable. The researchers distinguished proteins by size (1D) and charge (2D). Of course, just separating proteins is not enough, they have to go through

dyeing

. There are many choices of dyes, depending on the sensitivity requirements, existing equipment and downstream applications.

Conventional choice

Researchers mainly use three dyes to handle everyday

Protein gel

:

Coomassie Blue

, silver staining and fluorescence. According to Jeffrey Turner, senior research and development manager for protein technology at Sigma-Aldrich, the choice of dye can basically be attributed to "sensitivity vs. speed."

Coomassie blue is perhaps the most commonly used dye. As a visible color (bright blue) dye, it does not require any special equipment. However, Ning Liu, business development manager at Bio-Rad, believes that it has the lowest sensitivity and the detection limit is about 10-100 ng protein.

Many companies offer different formulations of Coomassie blue dyes that vary in ease of use, overall dyeing time, and discoloration requirements. For example, some need to be fixed in ethanol/acetic acid, while some use only pure water. Expedeon's InstantBlueTM reagent can be added directly to the gel after electrophoresis without the need for fixation or discoloration, and staining can be done in 15 minutes. Bio-Rad's QC Colloidal Coomassie Stain operation is recommended to take your time to achieve better results. The entire procedure consisted of fixing in 40% ethanol/10% acetic acid for 15 minutes, followed by staining for 1-20 hours and then bleaching for 1-3 hours.

With Thermo Fisher's latest PierceTM Power Stainer, researchers can automate Coomassie Blue staining. According to its product manager Emily Goplen, this device can dye and discolor two gels. “Although traditional dyeing takes hours or even overnight, the Power Stainer can complete the dyeing and bleaching process in 10 minutes, and both pre-formed and homemade glues are suitable,” she said.

Another common method is silver staining, with a sensitivity of approximately 0.1-0.25 ng per strip. There is no decolorization step in the operation, but a very long process, about 2 hours, and quite elegant. In addition, according to Turner, the operation requires the use of some harmful chemicals, such as formaldehyde and silver, preferably in a fume hood. Therefore, people are generally not willing to use this method unless sensitivity is important.

Fluorescent dyes are centrally sensitive and can detect a few nanograms of protein, but you have to have a compatible gel imaging system or transilluminator. Like Coomassie blue and silver staining, fluorescent staining is usually performed after electrophoresis and requires fixation and discoloration, with some exceptions. For example, SYPRO Ruby needs to be discolored, but Oriole and Flamingo are not needed because the free dye does not fluoresce. “The dye itself is not excited by the laser and is only excited when it binds to the protein,” explains Liu.

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