Characterization
At the Department of
Biochemistry we have the following
protein characterization tools:
It is assumed that the
protein has been well purified
and is pure on an
overloaded SDS-PAGE analysis. As a rule of thumb, the protein samples
obtained from his-tag affinity chromatography are not pure enough. It
is best to have at least one ion exchange column in the purification
protocol. The last purification step is always a size-exclusion
coulumn. The best crystallization results are obtained when using only
the central part of the peak fractions. Each characterization technique
has different
requirements for buffer, buffer concentration and protein
concentration. Consultation with the local staff is required to discuss
these issues, as they depend also on the properties of the protein to
be investigated. The X-ray
homepage also provides an extensive set of
bioinformatics links that should be consulted for characterizing the
protein before starting the experimental work. Knowledge of the
isoelectric point of the protein is important.
Differential scanning
fluorimetry (DSF) is a quick method to screen buffer
components and additives (buffer, ligand, additive and salt) which
increase the protein stability and thereby may improve the outcome of
the
protein purification and crystallization. At the Department of
Biochemistry we are using DSF to characterize protein samples. The
analysis is done in a 96-well format using a conventional real time PCR
machine utilizing an environmentally sensitive dye SYPRO orange which
binds to the hydrophobic interior of the protein when it undergoes
thermal denaturation. An increase in the fluorescence is measured when
the protein unfolds and the dye binds to the unfolded protein. Several
standard screens testing a range of
different pHs, buffers and salts are available for initial screening of
the thermal stability of the protein. The protein solution should be
around 0.5 - 1
mg/ml and in total about 400 μL of sample is minimally required. A new
user friendly interface has been developed to analyse the
data.
References:
Niesen FH, Berglund H,
Vedadi M. (2007) The use of differential scanning fluorimetry to detect
ligand interactions that promote protein
stability. Nat Protoc. 2, 2212-2221.
Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY,
Finerty PJ Jr, Wasney GA,
Yeung R, Arrowsmith C, Ball LJ, Berglund H, Hui R, Marsden BD, Nordlund
P, Sundstrom M, Weigelt J, Edwards AM. (2006) Chemical screening
methods to identify ligands that promote protein stability, protein
crystallization, and structure determination. Proc Natl Acad Sci U S A.
103, 15835-15840.
Ericsson UB, Hallberg BM,
DeTitta GT, Dekker N, Norlund P (2006) Thermofluor-based
high-throughput stability optimization of
proteins for structural studies. Anal Biochem. 357, 289-298.
To determine the molecular
weight, the oligomeric state and the
homogeneity (monodispersity) of the protein samples we have the Wyatt
MiniDawn static light scattering detector together with an
refractive index (RI) -detector (for the determination of the protein
concentration) connected to the ÄKTA purification unit. With this
method the absolute molecular weight is obtained witout the calibration
of the size exclusion-column. A small sample of 100 μL with a protein
concentration of 5 mg/ml is sufficient.
Circular dichroism (CD) is
a useful
tool to analyse the secondary
structure of the protein in solution. CD-melting curves give
qualitative information of the stability of the protein. At the
Department of Biochemistry we have the Jasco J-715 Spectropolarimeter
for CD measurements. For these studies the choice of buffer is
important. A phosphate buffer is a good buffer as it has no CD-signal
by itself. The protein concentration of the 400 μL sample should be
around 0.5 mg/ml. Cuvettes need to be cleaned immediately after use, in
particular in the case of melting curve measurements.
Additional information
There is much more
information available for the local users, on the Protein
Crystallography wiki, accessible to local users via the X-ray homepage.
Back
to X-ray Homepage
Updated August 19th,
2011
|
