Mutation of a predicted FurB binding site was achieved by inverse site directed mutagenesis PCR on plasmid pJEM- mptA2 using Phusion® polymerase and primers o mptA2-JEM-Mut-fw/rev with two single point mutations in the forward primer (mutated bases underlined, Additional file 1: Table S2) resulting in vector pJEM- mptA2MUT.
The saturation of unspecific binding sites is achieved by normal mouse serum control present in the staining buffer.
As with DC-SIGN, the clustering of these binding sites is achieved through an independent oligomerization domain that may provide some flexibility in the positioning of the binding sites.
Location of the ligand-binding site is achieved by matching numerical shape features describing the ligand to those of density clusters using a "fragmentation-tree" density representation.
The overall performance of STAT-Scanner was better than those of both MATCH and MotifLocator, as the detection of the same number of true binding sites was achieved by both with significantly lower total numbers of predicted sites.
Blocking of unspecific binding sites was achieved by incubation of the membrane in 5% low fat milk powder in PBS/0.2% Tween-20 (blocking buffer) for 1 hour at room temperature.
After the transfer, blocking of unspecific binding sites was achieved by incubation in TBST (50 mM Tris/HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.2) containing 5% skimmed milk.
Blocking of non-specific binding sites was achieved by incubating the membrane with 10% skim-milk in TBS buffer (10 mM Tris-Cl, 150 mM NaCl, pH 8.0) for 1 h.
Mutations of the Rbpj-binding sites were achieved by Fast Mutagenesis System (Stratagene).
More recently, the group also reported the design of a self-assembling Zn II -binding protein cryptand, in which a Zn II -bindingte is achieved in a structure temproteinby disulfide bonds.
However, the high potential for the Q/QH2 couple at both these Qi and Qo sites is achieved through preferential binding of QH2.
