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2-D Electrophoresis

The following protocols provide simple starting points for preparing crude protein extracts from common cell and tissue types.

Sample Protocol for Bacterial Cells

  1. Pellet approximately 109 cells in a 1.5 ml centrifuge tube (about the amount of cells from 1 ml of a 1 OD E.coli culture)
  2. Resuspend the cells in 40 ul of SDS/DTT lysis solution (0.3% SDS, 0.2M DTT, 50mM Tris-HCl pH 8.0)
  3. Heat at 95ºC for 5 minutes
  4. Place tube on ice and add 4 µl of DNAse/RNAse solution (50mM MgCl2 2units/ µl DNAse I, 0.75 units/µl RNAse A, 0.5M Tris-HCl, pH 8)
  5. Incubate on ice for 20 minutes
  6. Add sufficient urea/thiourea rehydration buffer (7M urea, 2M thiourea, 2% CHAPS, 10mM DTT, 2% Ampholytes pH 3-10, 0.01% bromophenol blue) to bring the total volume of sample to between 350 to 400 µl.
  7. Place the tube on ice and sonicate
  8. Incubate the sample at 30 ºC for 1 hour
  9. Centrifuge the sample at 20,000 x g for 10 min at room temperature and collect supernatant as protein extract.

Sample Protocol for Cultured Mammalian Cells

  1. Harvest, wash and pellet cells.
  2. Resuspend the pellet in urea/thiourea rehydration buffer (7M urea, 2M thiourea, 2% CHAPS, 10mM DTT, 2% Ampholytes pH 3-10, 0.01% bromophenol blue) to the desired concentration. (The amount of material required is dependent upon the cell type, pH range of the isoelectric focusing strip, the size of the gels, and the staining method. A suggested starting point for detection with fluorescent dye or silver is 2-5 million cells per large format pH 4-7 gel.)
  3. Sonicate the sample on ice.
  4. Incubate at 30 °C for 1 hour.
  5. Centrifuge at 20,000 x g for 10 minutes at room temperature and collect supernatant as protein extract.

For adherent cells:

  1. Wash cells with PBS.
  2. Add urea/thiourea rehydration buffer (7M urea, 2M thiourea, 2% CHAPS,10mM DTT, 2% Ampholytes pH 3-10, 0.01% bromophenol blue) to the desired concentration (see note above).
  3. Collect sample and follow steps 3-5 above.

Sample Protocol for Liver Tissue

  1. Add 0.5-0.7g liver tissue into 22.5ml urea/thiourea extraction buffer (7M Urea, 2M Thiourea, 2% CHAPS ,10mM DTT, 2% Ampholytes pH 3-10, 0.01% bromophenol blue, 1X protease inhibitor cocktail).
  2. Sonicate thoroughly on ice.
  3. Centrifuge lysate at 10,000 x g for 10 minutes at RT and collect supernatant as protein extract.
Additional Steps And Modifications For 2-DE Sample Prepartion
To reduce and alkylate prior to IEF
  1. Follow the appropriate protocol to obtain protein extract/solution.
  2. Add Tris base to bring pH to between 8.4 and 8.7.
  3. Add DTT to a final molarity of 20mM and incubate at RT for 30 minutes.
  4. Add dimethylacrylamide to a final molarity of 50mM and incubate at RT for 30 minutes.
  5. Bring solution to a final molarity of 40mM DTT to quench reaction.
  6. Samples are ready for 2-DE

Sample protocol for TCA precipitation

TCA precipitation can be used to concentrate proteins from dilute samples, to remove substances that interfere with isoelectric focusing, and/or as a prefractionation step prior to 2-DE.

  1. Follow the appropriate protocol to obtain protein extract/solution.
  2. Measure volume of supernatant and add 50% TCA (trichloro acedic acid) to yield a final TCA concentration of 10%.
  3. Incubate sample at –20ºC for 10 min.
  4. Centrifuge at 20,000 g for 5 minutes at 4ºC and discard supernatant.
  5. Wash pellet with 500 µl of 90% acetone and centrifuged at 20,000 g for 5 minutes at 4ºC and discard supernatant.
  6. Repeat step 5.
  7. Air-dry pellet until moist to remove excess acetone (do not overdry).
  8. Add 400µl urea/thiourea buffer ( 7M Urea, 2M Thiourea, 2% CHAPS ,10mM DTT, 2% Ampholytes pH 3-10, 0.01% bromophenol blue) to pellet and sonicate to solubilize.
  9. Centrifuge at 20,000 g for 5 minutes at room temperature and collect supernatant as protein extract.