Biology Reference
In-Depth Information
7. Dilution rate depends on the sample used. Determine dilution
rate of the sample if needed.
8. Calculate the volume of the solution needed based on the protein
concentration of the sample analyzed by the Bradford method.
9. To avoid contamination, gloves and a mask are necessary.
10. The following parameters are used for searches in MASCOT.
Database: NCBInr; Taxonomy: all entries; enzyme: trypsin;
number of missed cleavages: up to one missed cleavage; fi xed
modifi cation: carbamidometyl cysteine; variable modifi cation:
oxidation of methionine; peptide tolerance: below 50 ppm;
mass values: MH+. The protein is considered a positive candi-
date when it satisfi es all of the following three criteria: (1) sig-
nifi cant MASCOT score (
P
< 0.05); (2) more than four peptides
matched; and (3) sequence coverage of more than 15 %.
11. When candidate is not identifi ed in NCBI-nr, the barley gene
index (
http://compbio.dfci.harvard.edu/tgi/
)
and HarvEST
#31 (
http://harvest.ucr.edu/
)
, are appropriate using the same
parameters to attempt protein identifi cation.
References
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