Proteomics Core
Protocols
The standard protocols performed in Proteomics Core are listed below:
2-D Gel electrohporesis
To help you in your sample analysis, we have included web links to the companies
and Swiss Institute of Bioinformatics for the sample preparation and 2-D Gel
Electrophoresis guidelines.
From ExPASy Server (http://ca.expasy.org/ch2d/protocols/)
Bio-Rad http://bio-rad.com/
-Expression Proteomics http://www.expressionproteomics.com/
GE (Amersham Biosciences) http://www.gelifesciences.com/
The main Proteomics Core Facility (CSRB331) supports regular 2-D gels of Bio-Rad system for self-service. For Amersham’s Difference Gel electrophoresis (DIGE), please contact Mr. Guidry for details.
Staining Protocols
The Core Facility recommends Sypro Ruby and Colloidal Coomassie (Bio-Safe and Simple Blue) staining methods. The manufacturers’ protocols
are followed.
Protein Identification is achieved by measuring the tryptic peptides of proteins, obtaining partial sequence information, and searching the known databases to match possible protein matches.
Gel Handling
The major contaminations from gel samples are human keratins (from skin and hair) through handling. To reduce the keratins, usually use clean equipment and a fresh pair of gloves right before handling the gels. Also, try to grab the edge of the gel rather than the areas where the protein bands or spots are, during the gel transfers, staining, rinsing, etc. Minimize the gel exposed to dust from atmosphere.
In-Gel Digestion
Prior to the trypsin digestion, the gel plugs have to be destained/equilibrated with buffer. A blank gel plug is picked as control. The liquid handling robots will perform the destaining, in-gel trypsin digestion for 2D gel spots
Destaining Gel Plugs (for Coomassie and Sypro stains)
1. Working in a dust- free area, pick gel plugs from your protein of interest.
2. For one-dimensional gels, further slice the bands into 0.5-1 mm3 cubics (~0.75-1 mm each side). Place gel plugs in clear polypropylene tubes/96-well plates. For a two-dimensional gel pick as much of the spot as is practical without collecting too much background acrylamide.
3. Incubate gel plugs with 100 µl 25 mM ammonium bicarbonate in 5 % acetonitrile for 30 minutes. Remove the wash solution.
4. Incubate gel plugs with 100 µl 25 mM ammonium bicarbonate in 50 % acetonitrile for 30 minutes. Remove the wash solution. Repeat as needed in order to successfully destain plugs.
5. Incubate gel plugs with 100 µl 100% acetonitrile for 20 minutes. Remove acetonitrile solution. Dry gel plugs at 37°C for 10 minutes.
In-gel Trypsin Digestion
1. Prepare 10 µg/ml trypsin by adding 100µl of 50mM Acetic Acid (or
trypsin resuspension buffer from the manufacturer) to the lyophilized trypsin.
Add 900µl of 20 mM ammonium bicarbonate to make 1mL.
2. Add 8-20 µl trypsin solution to each tube to cover the gel plugs. Seal
plate/tube. Add few µl of 20 mM ammonium bicarbonate to cover the gel plugs
if 20 µl trypsin is not enough to over them.
3. Incubate gel plugs at 37oC for 3hours to overnight.
4. Add 50 µl 50% acetonitrile/0.1% TFA. Incubate at room temperature for 20
minutes. Remove extract solution and save.
5. Add 50 µl 50% acetonitrile/0.1% TFA. Incubate at room temperature for 20
minutes. Remove extract solution and combine with earlier saved extract solution.
6. Dry combined extract solution.
For 1D gel bands, the volume of the rinsing buffer and the amount of trypsin would be adjusted accordingly.
C18 ZipTip cleaning up
For rapid removal of excess salts/low M.W. impurities in mass spectrometry
samples. The ZipTip from Millipore is a 10-?l pipet tip packed with small
amount of C18 (for peptides) or C4 (for proteins) reversed phase chromatograph
media at the pointed end. The maximum capacity of the tip is typically 5 ?g.
Protocol can be downloaded at www.millipore.com/ziptip.
MALDI Spotting
1. Re-suspend the dried extracts in 2-5µl of 0.1% TFA with 0-50% acetonitrile.
2. Prepared a saturated solution of a-Cyano-4-hydroxy-cinnamic acid (10 mg/ml)
in 50% Acetonitrile and 0.1% TFA. The supernatant is the working matrix solution.
3. In a tube, mix well 1-2 µl of sample solution with 2 µl of
matrix solution. Immediately spot 0.7 µl of this mixture onto a MALDI
plate. Alternately, 0.5 µl of sample solution is spotted on the MALDI
plate and allowed to air dry. Then, 0.5 µl of matrix solution is spotted
on top of the sample spot.
The Proteomics Core Facility is located in the Clinical Science Research Building room 331. For further information please contact
Chau-Wen Chou, PhD (cchou@lsuhsc.edu).
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