The role of the buffer volume as an acoustic filter is investigated and optimized dimensions of the buffer volume, to achieve minimum noise transmission coefficient, are calculated.
After that, the loaded column was first subjected to washing with 10 column volume buffer A to remove the unbound protein fractions, then washed sequentially with buffer B (added 0.5 M NaCl in buffer A) to remove some stubborn protein, and then buffer A was used once again to lower the salinity.
The beads were then washed with 10 column volumes buffer E to remove MutSΔC800-MutSΔC800 crosslinks, after which the protein was eluted in buffer E with 300 mM imidazole and DTT was added to quench excess crosslinker.
An injection loop was then used to transiently pulse the tethered molecules with buffer containing either 100 or 400 mM NaOAc, followed by a sufficient volume of buffer to remove the injected salt.
One side or both sides of the membranes were incubated with an excess of purified enzyme in 10 mL of 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, pH 8.5, for 24 h at 4 °C and washed three times with an equal volume of buffer to remove unbound protein prior to use.
Plugs were rinsed with Tris EDTA (TE) buffer (10 mM Tris HCl, pH 8.0, 1 mM EDTA), then incubated at 37 °C, 2 h with gentle shaking in TE buffer containing 1 mM phenylmethylsulphonyl fluoride (PMSF, Fluka 93482, 0.1 M in ethanol) to inactivate Proteinase K. Finally, plugs were washed 3 × 2 h in ten volumes TE buffer to remove PMSF.
The column was then washed with 10 column volumes of buffer A to remove weakly bound proteins and proteins retained in the matrix.
After the cell lysate was incubated with beads and washed with HEPES buffer (25 mM HEPES/KOH, 100 mM KCl, pH 7.4) three times, DTT was added to a final concentration of 2 mM, and nutation was continued at 4 °C for another 30 min. The beads were then washed eight times with an excess volume of washing buffer to remove DTT.
Purified GAGs were injected onto the column (1mL/minute) and washed with 5 volumes of binding buffer to remove unbound molecules before bound GAGs were eluted using a continuous salt gradient (0 1M NaCl in binding buffer), 1.5mL fractions were collected at a flow rate of 1mL/min and elution measured at an absorbance wavelength of 256nm.
The column was washed with 10 volumes of column buffer to remove non- and weak binding proteins.
