G-Rich sequences can spontaneously fold into quadruplex structures; therefore, a complementary strand must anneal to the G-rich sequence prior to refolding to form duplex DNA.
RBCs were lysed with ammonium chloride buffer prior to staining.
Protease inhibitors were added to all buffers excluding the urea buffer prior to use.
Deglycosylated ovalbumin was buffer exchanged into aqueous AmAc buffer prior to CIP dephosphorylation.
For example, α-lactalbumin refolding with ultrafiltration achieved several-fold higher productivity and lower buffer consumption compared to refolding with the batch dilution method.
Guanidine hydrochloride supernatant was dialyzed against urea buffer for 4 h and pooled with urea supernatant followed by a slow sequential buffer exchange to refolding buffer (50 mM Tris HCl, pH 7.5, 50 μM ZnO Ac 2, 5 mM DTT).
The modified protein was first dialysed into Tris ⋅HCl buffer (50 m m, pH 7.4) containing urea (6 m) to remove excess modification reagents, followed by dialysis into the same buffer in the absence of urea to refold the enzyme.
Briefly, for a 1 L MEL5 TCR refold, 30 mg of MEL5 α-chain was incubated at 37°C for 30 min with 10 mM DTT and added to refold buffer at 4°C (50 mM TRIS pH 8.1, 2 mM EDTA, 2.5 M urea, 6 mM cysteamine hydrochloride, and 4 mM cystamine).
After the proteins were blotted onto a nitrocellulose membrane, they were renatured in 0.1 M tris buffer containing 0.2% tween to attempt to refold any conformational epitopes that were lost during SDS treatment.
For each renaturation sample, solutions containing 30 µg heavy chain plus or minus 42 µg β2m and/or 10 µg synthetic peptide were added to refold buffer and incubated at 4°C with stirring for roughly 40 hr.
