They include low stability of primary antibodies from probe conjugation process, alteration of binding properties, difficulty of probe conjugation to antibodies, high cost of reagents, increases of time and labors, and cross-over nonspecific binding of secondary probes.
Seven compounds were selected as the starting point for the further structure modification in considering binding activity, synthesis difficulty, and structure similarity.
If the two sets were the same, in highly expressed genes SR proteins would have difficulty binding exclusively to exonic ends, as all codons would be both translationally and splice optimal.
This may be explained, at least in part, by structural studies which have shown that Grb14 may have more difficulty binding to phosphotyrosine containing ligands due to the presence of a non-glycyl residue at the end of the BC loop and the lack of a P+3 binding pocket in the SH2 domain [ 25].
Kinase inhibitors typically target multiple kinases due to the high conservation of the ATP binding pocket, explaining the difficulties in identifying kinase inhibitors selective for EphA4.
This finding raises the possibility that the monoclonal anti-HMGB1 antibody used for tissue staining could have difficulties in binding to HMGB1 if the epitope was already occupied by deposited anti-HMGB1 antibodies.
Our work underscores the value of exploring water-mediated interactions to study ligand binding, and highlights the difficulty of optimizing polar interactions due to high desolvation penalties.
If the DNA template is crowded with trailing RNA polymerases, nascent RNA molecules will have difficulty in binding template DNA, disrupting R-loop formation (Fig. 1B).
To overcome this difficulty, TM binding to the open state was analyzed using the S1-induced TM binding assay in which actin saturation with TM under conditions unfavorable for TM binding alone was followed as a function of increasing S1 concentration (31, 38).
To overcome difficulties where the binding pocket conformation is unsuitable for docking, our group developed a new simple and fast approach to simulate the induced-fit conformational changes of protein structures upon binding.
