Our protein identification service is conducted
in-house. Thus, there is no time wasted shipping your samples
elsewhere for mass spec analysis after 2D-DIGE analysis
is completed. The turn-around time for protein identification
is 7-10 business days after spots are selected from the
gel.
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)
Call our tech support at 510-887-0889 or send email
to support@appliedbiomics.com
with any technical questions.
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