Biology Reference
In-Depth Information
the resolving gel (total migration time of about 50 min)
(short run, Fig.
2b
) (
see
Note 18
).
4. Stain the gels with colloidal blue or silver nitrate (
see
Note 19
).
5. For colloidal blue staining, wash gel three times 15 min with
water before staining overnight using the PageBlue™ Protein
Staining solution. Destain with water. Store gels in storage
solution S7.
6. For silver nitrate staining (
see
Note 20
), incubate gels over-
night in fi xation solution S1, then rinse threefold 10 min in
washing solution S2 and threefold 10 min in water. Perform
sensitization for maximum 2 min using solution S3. Rinse the
gels threefold 10 min in water. Carry out silver impregnation
30 min at 4 °C using solution S4. Rinse the gels threefold
10 min in water and once with washing solution S5. Achieve
development using solution S6. Stop staining with solution S7.
Store gels in storage solution S7.
3.6 Bioinformatic
Analysis of Proteins
Identifi ed by MS
1. Predict subcellular localization with different software like
TargetP, Predotar, and PSORT (
see
Note 21
). Each of them is
working with a different rationale. TargetP and Predotar are
neural-based tools trained on experimentally proven signal
sequences. PSORT is based on amino acid sequence proper-
ties, e.g. PSORT looks for a N-terminal basically-charged
region and a central hydrophobic region to predict a signal
sequences. Predict the presence of a GPI anchor site with
GPIsom and BigPI software.
2. Predict functional domains with PROSITE and PFAM.
PROSITE software can predict short patterns with a high
probability of occurrence as well as motifs and patterns (
see
Note 22
). PFAM predicts two types of domains: PFAM-A
entries are of high quality and are manually curated. PFAM-B
entries are automatically generated and only give indications
when no PFAM-A domain is found. InterProScan collects the
results of predictions of several software such as PROSITE and
PFAM and proposes IPR superfamilies grouping domains
predicted by these software. When no results are obtained with
these tools, look for proteins sharing common sequences
using the BLAST tool. In addition to proteins showing
sequence homology to the protein of interest, BLAST also
provides a library of conserved domains (CD).
3. Predict
N
-glycosylation sites and composition of
N
-glycans
attached to the predicted sites using MALDI-TOF MS data
with
ProTerNyc
.
ProTerNyc
uses (1) PROSITE, since
N
-glycosylation sites are short patterns of four amino acids
(N-{P}-[ST]-{P}, where N (Asn) is the
N
-glycosylation site) and
(2) literature data describing the structures of
N
-glycans [
33
].
