Biology Reference
In-Depth Information
3. Pay attention to the compatibility charts of the protein assay
used. High concentrations of SDS (>0.1 %) interfere with
Bradford, while DTT (>1 mM) interferes with BCA. The opti-
mal choice for protein quantification would be a reducing
agent-compatible BCA assay distributed by some suppliers.
4. The SDS-PAGE gel can be prepared in-house with standard
laboratory equipment. It is important to allow overnight
polymerization of the gels prior to use! Otherwise, unpolymer-
ized acrylamide will be cross-linked to peptides and prevent
peptide identification.
5. All solutions should be freshly prepared. Use HPLC-grade
H
2
O only.
6. To obtain a concentrated protein sample take maximal 10-20 μl
extraction buffer per mg fresh weight. Adjust protein concen-
tration to a value of 2 mg/ml.
7. It is advisable to minimize experimental variation and gather
the samples from all biological replicates to load them on the
same gel. This will minimize differences between the samples
that are due to unavoidable technical differences in the proto-
col application. If not all different biological replicates can be
applied on the same gel, alternative statistical tests such as
paired t-tests can be considered, which will help in uncovering
true differences between wild type and mutants despite the
variation introduced by gel-to-gel differences.
8. Avoid keratin contamination! Wear gloves and a lab coat at all
times during the experiment! Work in a clean and dust-free
environment! Do not lean over gels, tie long hair, and wear a
cap or a head cloth; do not wear clothes made from wool!
9. At each step gel pieces should be covered with the appropriate
solution. Do not autoclave pipette tips or solutions. Use
Eppendorf Safe Lock tubes.
10. Alternatively the tryptic digest can be performed at 37 °C for 4 h.
11. Take care not to confuse
spectral counting
with
peptide count-
ing
. Spectra that should be counted are referred to as peptide
spectrum matches (PSMs). PSMs are highly redundant and
many hundred spectra often identify a single peptide. It is this
redundancy and the repeated sampling of abundant peptides
and proteins that make spectral counting so robust for quanti-
fication purposes.
12. There is debate on how ambiguous peptides mapping to dif-
ferent protein sequences should be treated. While some col-
leagues suggest diverting the peptides between the different
proteins that they identify, or assign all peptide identifica-
tions to the highest scoring gene model, we decided to only
accept unambiguous peptide identifications in all our analyses.
