The GAD65 protein was dialysed against phosphate buffered saline (PBS) in order to remove mercaptoethanol in the storage buffer and aliquots (100nL) at a final concentration of 0.5 mg/ml GAD65 were then deposited onto the fresh pre-treated prism surface using the Hamilton starlet robot and a modified pin tool protocol that minimised contact of the pin tool with the gold coated surface.
The preparation of the conjugates themselves can be time-consuming since it requires a buffer transfer step to remove reducing agents (which are usually present in the storage buffer of a protein which contains free cysteines), following which the protein must be incubated with the conjugate for 1 or more hours, and then a 2nd buffer exchange must be carried out to remove unreacted conjugate.
Enzyme was kept in the storage buffer at pH 8 and diluted into assay buffers at the desired pH immediately prior to measurement.
The tests were carried out in the storage buffer of the FastStart® Taq DNA polymerase (Roche Applied Science, Mannheim, Germany), supplemented with 10 mM MgCl2, 1 mM CaCl2 and 1 mM DTT that are required by the dsDNases to be active or to be efficiently inactivated.
After the validation, the beads were counted, and stored in the storage buffer at 4°C.
Following a further 5 min, the isolated myocytes were resuspended and stored in the storage buffer.
The desired protein was then desalted and kept in the storage buffer (25 mM Tris-HCl, pH 8).
Addition of ethylene glycol to a final concentration of 1 M in the storage buffer was crucial to ensure the enzyme stability.
Especially the presence of Cl--ions in the storage buffer impaired the integrity of the radioimmunoconjugates, most likely due to the radiation-induced formation of OCl−-ions reacting with the SH-group of the enolised thiourea-unit.
This evidence might lead to the interpretation that long-range theta synchronizations induce the gating of relevant information in the storage buffer, as alpha oscillations have also been understood to be associated with the gating of sensory inputs (Worden et al., 2000; Payne & Kounios, 2009).
