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Looking for CRO to do protein Benzophenone Cross-linking, followed by Electroelution and Mass Spectrometry Analysis

  • Academic collaboration, Pharmaceutical process development and validation, Pre-clinical development and testing, pre-clinical CRO, Others
  • Biopharmaceuticals
  • Worldwide
  • 07/26/2012


Benzophenone Cross-linking
Protein to be reacted with a 10-fold molar excess of
benzophenone-4-maleimide (B4M) cross-linker (Invitrogen) in
the dark for 4 h in 100 mM Tris, pH 7.4, 0.1 mM EGTA, and 0.5
mM Tris(2-carboxyethyl)phosphine hydrochloride. The reaction
is quenched with 5 mM DTT and excess B4M is
removed using Nanosep centrifugal filter devices (Pall;
MWCO 30 kDa). B4M-labeled Protein (8 uM) is aggregated
overnight in 100 mM Tris, pH 7.4, 0.1 mM EGTA, and 5 mM
DTT in the presence of AA as described above. Cross-linking
Is induced with shortwave UV light (254 nm) for 5 min.
For sedimentation analysis, samples
are centrifuged in a TLS-55 Beckman rotor over a 40%
glycerol cushion at 269,000 X g for 30 min at 25 °C. The
supernatant is collected and the pellet is resuspended in
100 mM Tris, pH 7.4.

Cross-linked Protein aggregates are concentrated by sedimentation
(as described above), diluted in Laemmli buffer,
heated in a boiling water bath for 10 min, and separated by
SDS-PAGE using 4–8% linear gradient polyacrylamide gels.
Gels are stained with the E-Zinc Reversible Stain Kit (Thermo
Scientific) according to the manufacturer’s instructions. Individual
bands are isolated and placed inside D-Tube Dialyzers
(MWCO 6–8 kDa, Novagen). Samples are electroeluted for
5 h at 150 V in 25 mM Tris, 192 mM glycine, and 0.025% SDS.
Electroeluted proteins are concentrated to ~50 ul volume
with Pall Nanosep centrifugal concentrators prior to incubation
in 400 ul of SDS-away (Protea Biosciences) overnight at
-20 °C to precipitate the proteins. Samples are centrifuged at
18,000 X g for 10 min at 4 °C, the pellets are resuspended in
400 ul of SDS-away, recentrifuged, and finally resuspended in
50 mM HEPES, pH 7.6, 50 mM KCl, 0.01% Triton X-100. Samples
are boiled for 5 min and centrifuged at 16,000 X g for 1
min to remove insoluble proteins.

Mass Spectrometry Analysis
Protein samples are subjected to analysis by surface
enhanced laser desorption and ionization time of flight (SELDITOF)
mass spectrometry to examine the distribution of masses
of protein components. Samples are spotted in three successive
5-ul aliquots onto individual spots of a ProteinChip NP20
Array (Bio-Rad) and allowed to partially air dry for ~10 min to
reduce the volume between applications. The chip was then
washed three times with 5 ul of distilled water and air-dried
prior to the addition of 2.5 ul of 10 mg/ml sinapinic acid
(Bio-Rad) in 60% acetonitrile, 0.1% formic acid.
Samples are analyzed using a Bio-Rad ProteinChip System 4000 Enterprise
mass spectrometer calibrated against the Bio-Rad All-in-1-Protein
Standard. Each spot is divided into 10 partitions with 210 shots/partition
and the data are collected at a laser energy of 3000 joule with a mass
range between 10 and 160 kDa.

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