A method has been developed for extracting intact proteins from SDS-PAGE gels and analyzing them by MALDI-TOF MS to determine their molecular mass. This method consists of an electroelution step and a low-temperature matrix/analyte co-crystallization step prior to MALDI-TOF MS analysis. The source of this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillemment, M. Zivi, C. Damerweil, V. Mitchen, eds.
Operation method
Electroelution of intact proteins from SDS-PAGE gels and their MALDI-TOF MS analysis
Materials and Instruments
Electrophoresis Buffer Protein Staining Solution Microcentrifuge Tubes Move 3.1 Protein Detection For more product details, please visit Aladdin Scientific website.
Horizontal Gel Electrophoresis Instruments Electroelution Kits Vacuum Centrifuge Concentrators
In general, gel staining and destaining are very easy to perform. In this method, we used ElectroBlue dye instead of the fixative Caulophyllum Blue stain to develop the protein color (see Note 1). 
( 1 ) After SDS-PAGE electrophoresis, rinse the gel (8 cm X 10 cm) with 300 ml of Milli-Q water or deionized water three times for 10 min each time.
( 2 ) Stain the gel with ElectroBlue dye by soaking the gel (8 cm X 10 cm) with about 20 ml of staining solution and shaking slowly for 2 h (see Note 3).
( 3 ) Decolorize the gel by rinsing with 300 mL of Milli-Q water or deionized water until the desired gel background is achieved. Change the water every 30 min.
3.2 Electroelution
( 1 ) Place the electroelution tube vertically upward, add 0.8 ml of Milli-Q water or deionized water, and close the cap. Wait 10 min and check for leaks. Empty the tube (see Note 4).
( 2 ) Cut the protein bands from the stained gel with a clean, sharp, straight-edged razor, transfer the gel fragments to the electroelution tube with a pair of tweezers, add 1X electrophoresis buffer (0.8 ml) to the tube, gently close the cap, and carefully place the tube on the support tray of the electroelution instrument so that the current can pass directly through the two film windows (see Note 5 ).
( 3 ) Fill the electrophoresis tank with 1X electrophoresis buffer and place the support tray of the electro-eliminator into the tank, making sure that the electro-eliminator tubes are completely immersed in the electrophoresis buffer and that the film of each tube is perpendicular to the electric field.
( 4 ) 120 V electrophoretic wash for 2 h (see Note 6).
( 5 ) After electrophoresis, flip the positive and negative electrodes and electrophoresis at the same voltage for 30~60 s ( see Note 7) .
( 6 ) Remove the gel fragments from the electroelution tube with forceps. Gently close the cap and return the tube to the support tray.
( 7 ) Place the support tray in a 2 L beaker containing the desired buffer or acidified water (1% anthranilic acid). dialyze overnight at 4°C, changing the buffer once.
(8) After dialysis is complete, transfer the protein solution to a 1.5 mL microcentrifuge tube and concentrate to about 5 μL using a SpeedVac (see Note 8).
( 9 ) Mix the concentrated protein solution (1 μl) with the matrix solution (1 μl, erucic acid, 10 mg/ml in 30% acetonitrile) in a 150 μl microcentrifuge tube, cover tightly, and allow to co-crystallize the protein and matrix at 4°C overnight (see Note 9).
( 10 ) Gently uncap the microcentrifuge tube and remove the supernatant with a 2 μl pipette. Add 1 μl of matrix solution to the tube to resuspend the crystals, vortex and shake, and then dispense the crystals onto a MALDI plate (see Note 10).