Q: How do I submit a
sample?
A: Please
contact us via email using the following
adress: ms-service@zmbh.uni-heidelberg.de
Q: What buffer should my
sample be in when submitted?
A: We prefer the
samples to be already submitted as
Coomassie-stained SDS-PAGE gels. The
gels should be thouroughly destained in
water and delivered in a clean &
keratin-free container. Alternatively
samples can also be submited in Laemmli
buffer and will be run on pre-cast gels
provided by us (+20€ extra charge/per
gel). Sample for 1D electrophoresis
should not exceed a volume of 50 µl, for
2D electrophoresis the maximum is 125 µl
(not exceeding 50 ug of total protein).
On request and depending on the project
needs we can also perform in-solution
digestion employing either the widely
used "8M Urea protocol" or the "FASP
protocol" (see
Methods - Sample preparation). For
this, please contact us in advance for
details regarding sample buffer
composition, interfering substances etc.
!
Q: Are detergents in my
sample a problem? How can I remove them?
A: Most standard
detergents used in the laboratory (e.g.: SDS, Tween,
Triton, ...) are NOT
compatible with downstream
LC/MS-analysis and thus should not be
contained in your samples. Therefore, if
they cannot be ommited during sample
preparation they have to be removed. For
this, we recommend to precipitate your
protein(s) using the "Wessel-Fluegge
protocol" (see
Protocols). The resulting dried
protein pellet can then be processed by
us.
Alternatively, one could also use the
MS-compatible detergent RapiGest
SF sold by Waters
Corporation. For details regarding
this option please contact us.
Q: How much protein is
necessary for identification or
quantification?
A: In general
Coomassie stained gel bands have a high
success rate for identification. We
recommend to load up to 50 ug of
proteins per well for a complex sample
mixture (total amount) and a minimum of
50 ng of a single protein band.
Q: Can I submit silver
stained gels?
A: Generally,
Silver-staining is not the best method
to combine with mass spectrometry (see
also the considerations made in
"Laborjournal"- in German only).
We rather recommend using sensitive
colloidal Coomassie blue stains. But
note that shortly developed silver
stained gels, for which mass spec
compatible reagents were used, can also
be submitted. For this, the SilverQuest
Silver staining kit from ThermoFisher
Scientific (Cat.nr: LC6070) is
preferred.
Q: Is a specific 1D gel
system or Coomassie-stain recommended?
A: Most SDS-PAGE
systems are suitable for downstream mass
spectrometry. We can recommend 4-12%
gradient NuPAGE Bis-Tris precast gels
(ThermoFisher Scientific preferred) as
they offer excellent separation and
sensitivity together with a colloidal
Coomassie blue stain (ThermoFisher
Scientific or SERVA Quick Stain
preferred).
Q: When can I expect my
results back?
A: We process
the samples according to "first come
first serve" and aim for data return
within two weeks. However this might
vary, depending on the total sample
number in the queue as well as the
complexity of the workflow. Thus, an
individual approximation of the
processing time will be given during
sample submission.
Q: What possibilities are
there for quantification?
A: Possibilities
include label free (MaxQuant LFQ,
Spectral counting), chemical (e.g.
Dimethyl) or metabolic labeling (e.g.
SILAC) as well as absolute labeling
using AQUA peptides.
Q: What are the first
steps for SILAC? Where do I get the
labeling media?
A: Cellular
isotope labeling has to be done by you,
the user. The required SILAC media can
be obtained from various suppliers e.g.
Silantes,
ThermoFisher
Scientific etc.. To obtain
reliable SILAC-quantifications we highly
recomend to check the incorporation
efficiency of the heavy amino acids
within your cell line of choice before
starting any the real experiments. Note
that we have successfully performed
SILAC on E.coli, yeast and
various mammalian cell lines like
HEK-293, HeLa and microglial cells.
Q: What do you charge?
A: You can find
our current price list under "Protocols".
For a more specific quote adapted to
your project, please contact us by
email.
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