Proteomics

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Proteomics
Proteomics is the large-scale study of proteins. The facility has several high-resolution mass spectrometers for proteomics analysis, including Orbitrap mass spectrometer as well as Q-TOF LCMS with microflow LC system for high sensitivity. These high-end capabilities enable researchers to dig deeper while targeting low-abundance proteins and identify more proteins at faster time scales – leading to more accurate and thorough quantification. The facility has high end labeled (e.g. iTRAQ, TMS etc.) as well as label free (SWATH) quantification workflows for relatively quantified proteins among several groups for biomarker discoveries. The facility also develops methods for quantification of targeted proteins in given samples.
 
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Resources for Proteomics

Methanol/Chloroform Precipitation Method

1. Add equal volume (100 uL) of 100% methanol and 62.5 uL of chloroform to 100 uL of soluble Protein and mix well for 5 min.

2. Centrifuge at 12,000_ g for 15 min.

3. Remove supernatant without attaching the interface layer (protein fraction) by pipette.

4. Add 100 uL of 100% methanol to the sample and mix gently for 5 min. Centrifuged at 12,000_ g at 25 _C for 15 min.

5. The supernatant is discarded, then the pellet air-dried.

Acetone Precipitation Method

1. Cool the required volume of acetone to -20°C.

2. Place protein sample in acetone-compatible tube.

3. Add four times the sample volume of cold (-20°C) acetone to the tube.

4. Vortex tube and incubate for 4 hours at -20°C (can be kept overnight).

5. Centrifuge 10 minutes at 13,000-15,000 × g.

6. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet.
Optional: If additional cycles of precipitation are necessary to completely remove the interfering substance, then repeat
steps 2-5 before proceeding to step 7.

7. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do not over-dry pellet, or it
may not dissolve properly.

8. Add buffer appropriate for the downstream process and vortex thoroughly to dissolve protein pellet.