General principles:

Proteins in the samples must be fully denatured, reduced, and solubilized for effective IEF separation. Samples are rapidly lysed in a strongly denaturing solution with brief sonication in the ice container. Protease inhibitors can be added in the lysis solution to prevent protein degradation. Proper conditions for lysis vary greatly depending on the source of the samples, and should be determined empirically.

A minimum of 25 ug total protein, regardless of the source of samples, is required for analytical 2-D DIGE. Depending on the abundance of the target protein, at least 200 ug total protein may be needed for prep-DIGE gel, spot picking and mass spectrometric analysis. Please submit enough sample protein so that we can complete your project successfully. We can perform the standard sample prep steps for the customers. For the plasma and serum sample immuno-depletion, additional charge will apply. Please provide us with specific instructions if special handling is required. If you wish to lyse the samples yourself, please adjust the final concentration of total protein to 3-20 mg/ml. Please contact us for the appropriate lysis buffer to use for your samples.

The following are the basic guidelines for protein lysate preparation:

1. It is essential that lysis buffer is free of both primary amines and sulfhydryl groups.
2. Follow the sample preparation protocol described in GE Healthcare's DIGE section.
   
   

   Not Acceptable	Acceptable
   Reducing agents (e.g., DTT, TBP) Buffers other than Tris Salt (e.g., NaCl) Ionic detergents (e.g,. SDS) Nucleic acids Polysaccharides Lipids Phenolic compounds Insoluble material Other small ionic molecules	Tris base (< 30 mM) Urea Water Non-ionic or zwitterionic detergents (e.g. CHAPS)

   
   

Sample preparation based on sample sources:

Customers may consider the following sample preparation modifications to achieve optimal results.

Laser Capture Micro-dissection (LCM) capture to separate tumor cells from normal cells in the same tissue sample
FACS or Cell sorting to separate different cells (cell cycle stage or cell types)
Protein Complex isolation and sample fractionation
Cell surface membrane protein isolation
Mitochondria isolation, ER, nucleus, plasma, protein fractionation
Pre-isolating the specific cells of interested

1. Bacteria. Bacterial cells are pelleted by centrifugation. Pellets are washed twice with cell washing buffer (10 mM Tris HCl, 5 mM magnesium acetate, pH 8.0). After the final spin, the supernatant must be removed as completely as possible. Cell pellets are quickly frozen in liquid nitrogen or dry ice/ethanol bath. Frozen cell pellets can be shipped to us on dry ice.
   
   
2. Yeast. Yeast cells are pelleted by centrifugation. Pellets are washed twice with cell washing buffer (10 mM Tris-HCl, 5 mM magnesium acetate, pH 8.0). After the final spin, the supernatant must be removed as completely as possible. Cell pellets are quickly frozen in liquid nitrogen or dry ice/ethanol bath. Frozen cell pellets can be shipped to us on dry ice.
   
   
3. Mammalian cell lines. For suspension cultures, cells are pelleted by centrifugation. Pellets are washed twice with PBS or cell washing buffer (10 mM Tris-HCl, 5 mM magnesium acetate, pH 8.0). After the final spin, the supernatant must be removed as completely as possible. Cell pellets are quickly frozen in liquid nitrogen or dry ice/ethanol bath. Frozen cell pellets can be shipped to us on dry ice.

   For adherent cultures, the cell monolayer can be washed twice with PBS or cell washing buffer (10 mM Tris-HCl, 5 mM magnesium acetate, pH 8.0). After the second wash, a small volume of cell washing buffer is added to the plates (1 ml for each 100-mm plate), and cells are scraped from the plates. The scraped cells are transferred to eppendorf tubes, and cells are pelleted by a 1-minute spin at low speed. Remove the supernatant as completely as possible. Cell pellets are quickly frozen in liquid nitrogen or dry ice/ethanol bath. Frozen cell pellets can be shipped to us on dry ice.

4. Enriched cell organelles (nuclei, mitochondria, membrane fraction, etc.). Following the purification steps, wash the enriched cell organelles twice with cell washing buffer (10 mM Tris-HCl, 5 mM magnesium acetate, pH 8.0). If necessary, neutral substances (such as sucrose) may be included in the washing buffer. After the final wash, the supernatant must be removed as completely as possible. Pellets are quickly frozen in liquid nitrogen or dry ice/ethanol bath. Frozen pellets can be shipped to us on dry ice.
   
   
5. Purified protein complex. After immunoprecipitation, wash the protein complexes twice with cell washing buffer (10 mM Tris-HCl, 5 mM magnesium acetate, pH 8.0) to get rid of salt and ionizing detergents. If this causes a problem, please contact us. Usually, cross-linked antibodies (to the protein A/G beads) give better background. We will design a protocol based on your special requirements. Send the washed pellets to us on dry ice.
   
   
6. Animal tissue. About 5-10 mg of animal tissue (approximately 2 mm x 2 mm x 2 mm in size) is needed for 2-D DIGE analysis and the mass spectrometric protein analysis. We suggest that the contaminating blood be removed using PBS. Tissues can be flash frozen in liquid nitrogen after harvest. The tissue is then ground to a frozen powder in a mortar and pestle. Frozen tissue powder can be shipped to us on dry ice.
   
   Tissues that are embedded in OCT blocks are fully compatible with 2-D DIGE analysis. Embedded tissues can be shipped to us on dry ice.
   
   
7. Human tissue. . About 5-10 milligrams of human tissue (approximately 2 mm x 2 mm x 2 mm in size) is needed for 2-D DIGE analysis. Removing the contaminating blood by washing with PBS is recommended. Tissues can be flash frozen in liquid nitrogen after harvest. The tissue is then ground to a frozen powder in a mortar and pestle. Frozen tissue powder can be shipped to us on dry ice.
   
   Tissues that are embedded in OCT blocks are fully compatible with 2-D DIGE analysis. Embedded tissues can be shipped to us on dry ice.
   
   
8. Laser captured micro-dissected (LCM) cell population. A minimum of 20,000 laser captured micro-dissected cells is required for 2-D DIGE analysis. After LCM, adherent cells are eluted from the caps with small amount (10-15 l per cap) of lysis buffer. Lysates are collected (and pooled if more than one cap) in microcentrifuge tubes, frozen in liquid nitrogen or dry ice/ethanol bath. Frozen lysates can be shipped to us on dry ice and we will complete the lysis step.