- Staining Occurs During the Run
- Ideal Prep for Mass Spec
- 10ng Sensitivity in 30 Minutes!
The Insite System contains sufficient material to stain 60 mini-gels.
The Insite System is an in-gel fluorescent stain for proteins, providing excellent sensitivity (10ng) more quickly than any other detection method. The Insite stain runs with the proteins during electrophoresis. No staining step is required after electrophoresis. To use the Insite System, researchers simply mix Insite Buffer Concentrate with Insite Dye Concentrate and QS with deionized water to formulate their cathode tank buffer for SDS-PAGE. The Insite dye runs with the proteins during electrophoresis. After a short destain in water following electrophoresis, the bands are visualized under UV transillumination.
Proteins stained with the Insite System may be easily and reproducibly recovered at high yield from SDS-PAGE gels for analysis by mass spectroscopy.
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| The image on the left above shows the mass spec of Carbonic Anhydrase. Above right, the image shows the mass spec of Carbonic Anhydrase that has been electrophoresed by SDS-PAGE, visualized by the Insite System, and Purified from the Gel Slice by Crush and Soak Technique. |
Insite Protocol
Procedures for Using Insite:
1) Assemble gel apparatus.
2) Prepare 150 ml of Insite Detection Reagent by diluting 15 ml of 10X Insite Buffer Concentrate to 149 ml with deionized water and adding 1 ml of Insite Dye Concentrate. This solution has a useful life of about 3 hours.
3) Fill the anode buffer chamber with 1X Tris Glycine SDS (EC-870) (0.192M Glycine, 0.025M Tris, and 0.1% SDS). Fill the cathode buffer chamber with the Insite Detection Reagent.
4) Load the samples.
5) Run the gel under standard voltage and temperature conditions. Typical running conditions are 175 volts for 1 hour.
6) Remove the gel from the apparatus. [The gel may be observed immediately using a UV transilluminator (see below) for the detection of bands containing more than 300ng of protein.]
7) Destain the gel in 50 ml deionized water for 15 minutes. Repeat the wash in fresh deionized water for another 15 minutes. Alternative Destain: Destain the gel in 50 ml of 0.1M potassium chloride for 30 minutes. Using potassium chloride gives a lower, more uniform background, preserves staining for a longer period, and gives sharper bands.
8) Observe the gel on a transilluminator under ultraviolet illumination (302nm). Protein bands fluoresce yellow-orange. If excessive background is observed, wash the gel in an additional deionized water bath for 5-10 minutes.
9) Bands may be photographed using the #8 yellow filter used for coomassie stains or the #15 orange filter used for ethidium bromide. Note that background will increase as the gel is allowed to stand and the surface dehydrates. This background is easily removed by a brief wash in deionized water.