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Protein
Identification |
Download MALDI-TOF
Service Information
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You provide
us with a,b, or c:
a)
Homogenous protein in an excised band from a dried SDS-gel (stained
with Coomassie Blue or Silver, preferably without fixation). Please
enclose a gel photo. Please mention the %age of the gel, clearly
mark the band which was excised from the gel, and specify the
molecular weight marker and the estimated molecular weight of the
protein of interest.
b)
Homogenous protein in an excised spot from a dried 2-D-Gel (stained
with Coomassie Blue or Silver, preferably without fixation). Please
enclose a gel photo. Please clearly mark the spot which was excised
from the gel.
c) At
least 5 µg purified native protein (ammonium sulfate precipitate or
lyophilizd protein). Please enclose a gel photo (SDS-PAGE), mention
the %age of the gel, clearly mark the band which corresponds to the
purified protein, and specify the molecular weight marker and the
estimated molecular weight of the protein of interest. Recommend a
suitable buffer for dialyzing (ammonium sulfate precipitate) or
dissolving (lyophilized material) the protein. If this is not a
standard buffer, add your buffer in sufficient amount (esp. for
dialysis). Mention special requirements of your protein with respect
to solubility and stability (especially important for membrane
proteins).
Required
Information:
Source of the
protein (organism, tissue, cell fraction etc.) and, if possible, any
assumption about the nature and/or function of the protein of
interest.
If available: Molecular Weight of protein and its subunits, and
isoelectrical point.
Please ship the
protein without cooling (dried gel bands or spots, lyophilized protein)
or on blue ice (ammonium sulfate precipitate) to:
Gentaur bvba
Avenue de l'Armée 68/B4,
B-1040 Brussels
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Our service:
- (In gel)
trypsination of the protein (band or spot) of interest (protein is
cleaved at arg and lys sites into peptides)
- MALDI-TOF Mass
Spectometry analysis of the tryptic peptides
- Protein
identification by computational tools (database searching combined
with Bayesian statistics): Masses of peptides from the proteolytic
digestion of the corresponding protein are compared to the masses of
a peptide database based on the non redundant information extracted
from NCBI, GenBank, CDS translations, PDB, SwissProt, PIR, and PRF
protein databases. The result is a ranking of candidate proteins
based on their calculated posterior probability. The protein
identity is re-verified using pI, MW and peptide mass fingerprinting
data and comparing them with the theoretical peptides calculated for
all proteins of question in the SWISS-PROT/TrEMBL databases. This
verification process is carried out by trained scientists who verify
the protein identity by checking the peptide pattern peak by peak
for plausibility taking trypsin autolysis and trypsin cleavage
faults into account.
- You receive a
detailed analysis report featuring the identity of the protein and
the likelihood of the correctness of the identification. In case it
is a new protein, the features of similar/related proteins are
listed.
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Order
Information:
Cat. Nr.
PIPB01: Protein Identification by MALDI-TOF-MS, protein shipped
in protein gel band
Price: 500 Euro
Cat. Nr.
PIPS01: Protein Identification by MALDI-TOF-MS, protein shipped
in 2 D gel spot
Price: 500 Euro
Cat. Nr.
PIPP01: Protein Identification by MALDI-TOF-MS, purified protein
provided
Price: 570 Euro
Please note:
In case the protein amount or purity was not sufficient to perform
MALDI-TOF MS, a set up fee of € 175 is charged.
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Examples: |
The
SDS-PAGE gel illustration below shows the results of a protein
purification (upper band) by metal-chelate chromatography after trying
different washing conditions applied to eliminate the lower band
(impurity). |
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The
MALDI-TOF MS spectrum of the protein preparation confirmed the results
obtained with the SDS-PAGE. |
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The contaminant
has a molecular weight of 37095 Da. To efficiently eliminate the
impurity, the 37 kDa protein was identified by MALDI-TOF MS of a tryptic
protein digest:
The lower band was
excised from the gel and the in gel-digestion was carried out with
trypsin. The tryptic digest was analysed with MALDI-TOF MS. The
resulting mass spectra are given below:
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Protein Candidates |
Rank
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Probability
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Protein Description |
MW (kDa)
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1
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1.0e+00
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gi|6324486|ref|NP_014555.1| Alcohol dehydrogenase; Adh1p [Saccharomyces
cerevisiae] |
37
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2
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3.2e-03
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gi|1168350|sp|P00330|ADH1_YEAST ALCOHOL DEHYDROGENASE I |
37
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3
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7.8e-09
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gi|6324174|ref|NP_014244.1| Ynl155wp [Saccharomyces cerevisiae] |
32
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4
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1.1e-09
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gi|7245674|pdb|1YLV|A Chain A, Schiff-Base Complex Of Yeast
5-Aminolaevulinic Acid Dehydratase With Laevulinic Acid |
38
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5
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3.9e-10
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gi|171974|gb|AAA34790.1| (M81696) mitochondrial ribosomal protein
[Saccharomyces cerevisiae] |
29
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6
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3.2e-10
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gi|6325350|ref|NP_015418.1| Ypr093cp [Saccharomyces cerevisiae] |
36
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7
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3.0e-10
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gi|223142|prf||0512226A dehydrogenase isozyme I,alcohol [Saccharomyces
cerevisiae] |
32
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8
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3.0e-10
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gi|6320613|ref|NP_010693.1| 263-amino acid mitochondrial ribosomal large
subunit protein; similar to L23 family of ribosomal proteins; Mrp20p
[Saccharomyces cerevisiae] |
31
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9
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1.2e-10
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gi|626218|pir||S45958 probable membrane protein YBR090c - yeast
(Saccharomyces cerevisiae) |
13
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10
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1.2e-10
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gi|6319560|ref|NP_009642.1| mitochondrial ADP/ATP translocator; Aac3p
[Saccharomyces cerevisiae] |
33
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The contaminating protein was identified as alcohol dehydrogenase and
could now be removed easily from the mixture by exploiting its known
hydrophobicity (washing the column with isopropanol). |
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Our service:
Sample preparation
and MALDI-TOF Mass Spectrometry of the protein. Determination of the
absolute mass of the protein subunit(s).
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Order
Information:
Cat. Nr. DMW01
Absolute Molecular Weight Determination of purified protein by MALDI-TOF
MS
Price: 225 Euro
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send mail to [email protected] with questions or comments about this web site.
Copyright © 2008 Gentaur Molecular Products
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Last modified:
05/19/16
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