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 2-D Gel Electrophoresis manual (http://biorad.com)(PDF attached)
Bio-Rad’s general proteomics protocols (http://www.proteomeworks.bio-rad.com)
Amersham Biosciences (http://techsupport.apbiotech.com/)
(PDF attached)
The main Proteomics Core Facility (MEB 7112) supports regular 2-D gels of
Bio-Rad system. We offer service of running IGP strips at the sizes of 7 cm,
11 cm and 18 cm, and Mini-Protean 3, Criterion and Protean Plus PAGE, respectively.
All gels (IGP strips and PAGE) are pre-cast by Bio-Rad. Also, we stock 11
cm IPG (pH 3-10, and 4-7) and Criterion 8-16% gradient gels for user to run
their own 2-D gels. For Amersham’s Difference Gel electrophoresis (DIGE),
please contact Mr. Guidry for details.
Staining Protocols
The Core Facility provides the choices of Sypro Ruby, Copper, Coomassie (Bio-Safe)
and Silver (SilverPlus) staining methods. The manufacturers’ protocols
are followed.
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 and MALDI sample spotting
based on these protocols.
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 ?l 25 mM ammonium bicarbonate in 5 % acetonitrile
for 30 minutes. Remove the wash solution.
4. Incubate gel plugs with 100 ?l 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 ?l 100% acetonitrile for 20 minutes. Remove
acetonitrile solution. Dry gel plugs at 37°C for 10 minutes.
Destaining Gel Plugs (for silver stains)
1. Prepare a fresh working solution by mixing 30 mM potassium ferricyanide
(K3[Fe(CN)6]) and 100 mM sodium thiosulfate (Na2S2O3) stock solutions in a
1:1 (v/v) ratio.
2. Cover the gel plugs with the working solution. Incubate until the stain
disappears with occasional vortexing.
3. Rinse gel plugs with three changes of 100 ?l 25 mM ammonium bicarbonate
(NH4HCO3), with occasional vortexing.
In-gel Trypsin Digestion
1. Prepare 10 ?g/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 ?l trypsin solution to each tube to cover the gel plugs. Seal
plate/tube. Add few ?l of 20 mM ammonium bicarbonate to cover the gel plugs
if 20 ?l trypsin is not enough to over them.
3. Incubate gel plugs at 37oC for 3hours to overnight.
4. Add 50 ?l 50% acetonitrile/0.1% TFA. Incubate at room temperature for 20
minutes. Remove extract solution and save.
5. Add 50 ?l 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.
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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