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Proteomics Sample Preparations

 

 

1. We perform customized sample preparation using our proprietary protocols that best fit your specific sample type.

2. Biohazard materials: We do NOT accept samples containing any biohazard material. If your samples contain any biohazard materials, please make sure to deactivate them completely using the appropriate procedure and declare them in the order form. In addition, we do NOT accept any samples containing Level 4 biohazard materials even if they are deactivated. Please refer to the following link for the rank of biohazard materials:
http://en.wikipedia.org/wiki/Biological_hazard.

3. Please follow the general principles in getting your samples ready.

4. Please click on your sample type for additional tips:

Bacteria Animal tissue Mammalian cell lines
Yeast Human tissue Purified protein complex (IP samples)
Enriched cell organelles (nuclei, mitochondria, membrane fraction, etc.)
Laser captured micro-dissected (LCM) cell population

General principles:

For effective IEF separation, proteins must be solubilized, fully denatured and reduced. Protein samples can be rapidly lysed in a 2D lysis buffer containing strong denaturing reagents and protease inhibitors (optional). Proper lysis buffer can vary greatly depending on the source of the samples, and should be determined empirically.

Please feel free to submit samples in whichever buffer that you would normally use. The standard sample preparation is covered by the DIGE gel cost. Such samples should have protein concentration in the range of 5-20 mg/ml. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.

For gel-based proteomics studies, about 25 - 30 ug total protein, is required for analytical 2D DIGE. Depending on the  abundance of the  target protein, 200 - 300 ug total protein will be used for preparative 2D DIGE, spot picking and mass spectrometry analysis. For liquid-based proteomics studies, the required protein amount varies greatly depending the specific services, please contact us for the information.

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

Immuno-precipitation (IP) to purify protein complex
Fractionation of cell surface membrane proteins
Fractionation of Mitochondria, ER, nucleus and other cell organelles
FACS or Cell sorting: separate different cells (cell cycle stage or cell types)
Laser Capture Micro-dissection (LCM): isolate tumor cells from normal cells in the same tissue samples

If you want to use the lysis buffer that are compatible with IEF, please make sure that lysis buffer is free of both primary amines and sulfhydryl groups, and follow these guidelines 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)

 




  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. 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.

  4. Enriched cell organelles (nuclei, mitochondria, membrane fraction, etc.). Following the purification steps, the enriched cell organelles are washed 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. If you use IP in getting protein complex, please do the IP and washing procedure the same way you did before. At the end, please transfer the beads to a 1.5 ml eppendorf tube, wash the beads once with 0.5 ml of HPLC-grade water. Please remove the water as completely as possible after the final wash. The IP bead pellet can be kept at -20oC and sent to us on dry ice. We will elute protein complex from the beads using appropriate buffers.

  6. Animal tissue. About 5-10 mg of animal tissue (approximately 2 mm x 2 mm x 2 mm in size) is required for 2D DIGE / mass spectrometry analysis. Please remove the residual blood with PBS. Tissues can be flash frozen in liquid nitrogen after harvest, or directly stored at -80oC freezer. If needed, the tissue is then ground to a frozen powder in a mortar and pestle. Frozen tissues can be shipped to us on dry ice.

    Tissues that are embedded in OCT blocks are fully compatible with 2D DIGE analysis. Embedded tissues can be shipped to us on dry ice.

  7. Human tissue. About 5-10 mg of animal tissue (approximately 2 mm x 2 mm x 2 mm in size) is required for 2D DIGE / mass spectrometry analysis. Please remove the residual blood with PBS. Tissues can be flash frozen in liquid nitrogen after harvest, or directly stored at -80oC freezer. If needed, the tissue is then ground to a frozen powder in a mortar and pestle. Frozen tissues can be shipped to us on dry ice.

    Tissues that are embedded in OCT blocks are fully compatible with 2D 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 are required for 2D DIGE / mass spectrometry analysis. After LCM, adherent cells are eluted from the caps with small amount (10-15 ul per cap) of lysis buffer. Lysates are collected (and pooled if more than one cap) in eppendorf 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.