1. Gel treatment: 2D gel spots are
washed multiple times to remove staining dye and other inhibitory
chemicals. Gel spots are then dried to absorb maximum volume
of digestion buffer.
2. In-gel trypsin digestion: Dried 2D gel spots are
rehydrated in digestion buffer containing sequencing grade
modified trypsin. Proteins are digested in-gel at 37°C.
3. Peptide extraction: Digested peptides are extracted
from gel with TFA extraction buffer and shaking.
4. Desalting: The digested tryptic peptides are desalted
using C-18 Zip-tips (Millipore).
5. Spotting: The desalted peptides are mixed with
CHCA matrix (alpha-cyano-4-hydroxycinnamic acid) and spotted
into wells of a MALDI plate.
6. MALDI-TOF: Mass spectra (MS) of the peptides in
each sample are obtained using an Applied Biosystems Proteomics
Analyzer.
7. MALDI-TOF/TOF: Ten to twenty of the most abundant
peptides in each sample are further subjected to fragmentation
and tandem mass spectrometry (MS/MS) analysis.
8. Database search: Protein identification is based
on peptide fingerprint mass mapping (using MS spectra) and
peptide fragmentation mapping (using MS/MS spectra). Combined
MS and MS/MS spectra are submitted for database search using
GPS Explorer software equipped with the MASCOT search engine
to identify proteins from primary sequence databases.
9. Data Report: Includes the top 10 highest scoring
hits from the database search for each 2D gel spot, as well
as a summary listing the best match for each sample (see
a sample
report of protein ID analysis)
***********************************************************************************************************************
Guidelines
for Preparing and Shipping Samples for Protein Identification
1-D gel band:
Please keep in mind that a band from a 1D gel could contain
several proteins, although it looks a single band. This
can reduce the real amount of your protein of interest.
Low quantity and high complexity of samples will greatly
reduce the chance of success for high confident identification.
Please follow the tips below:
1. Protein amount. Our general rule
of thumb is that if there is enough protein to be strongly
stained with Coomassie G-250, there should be enough for
Mass Spectrometry analysis. We prefer that you scale up
your prep so that you have 10-20 ng of protein in the gel
band. Please do not scale up by sending multiple gel pieces,
as this increases gel volume and decreases the efficiency
of peptide extraction from the gel. Instead, load a higher
amount of protein into ONE lane of the gel.
2. Minimize gel volume. It is important
to minimize the gel volume and maximize protein concentration.
As such, we recommend you cut out only the area with your
protein of interest (1x1x2mm size) and exclude as much unstained
gel as possible.
3. Silver stained gel band should be compatible
with Mass Spectrometry. You need to make sure your silver
staining solution does not contain aldehyde that cross-links
your proteins to the gel matrix, resulting in low extraction
efficiency. Non-formaldehyde Silver Stain uses carbohydrazide
instead of formaldehyde for reduction/development.
4. Shipping condition. When you are
ready to send your sample, please add HPLC-grade water to
cover the excised gel band in a 1.5ml eppendorf tube, or
96 well plate. Seal the Eppendorf tubes with Parafilm. Put
the Eppendorf tubes into 50 mL conical tubes, then pack
the 50 mL tube with Kimwipes to prevent the Eppendorfs from
being crushed or jostled. The samples can be shipped on
ice pack (no need to send on dry ice) to the following address:
Proteomics Service
Applied Biomics, Inc.
23785 Cabot Blvd, Suite 313
Hayward, CA 94545
USA
5. Order form. Fill out the order
form 
.
The completed form can be included in the package, or faxed
or emailed to us.
Call our tech support at 510-887-0889 or send email
to support@appliedbiomics.com
with any technical questions.
Different Staining
of 2D Gels
download
the order form
