Briefly, 40 μL of the TTR variants at 4 μM (in GF buffer) was treated with 4 μL of glutaraldehyde (25% solution, Sigma) and incubated at room temperature for 5 min. The reactions were quenched by addition of 4 μL of 7% NaBH4 (freshly prepared in 0.1 M NaOH).
For multiplex analysis detection, the nanocomplex of 1 nM in PBS buffer was treated with the Tubb3 targets (single-base mismatched or complementary DNA targets) or Fox3 targets (single-base mismatched or complementary DNA targets) at various concentrations of 0, 2, 5, 10, 25, 50, 100, and 200 nM.
One third of the lysate in Passive Lysis Buffer was treated with 200 µl Trizol reagent and chloroform to extract RNA for reverse transcription (RT -PCR.
A blank control was added that contained only the extraction buffer and was treated in the same manner as the samples.
Total RNAs were isolated from the proliferating leaf explant cultures of M. truncatula line 2HA and Jemalong using the Qiagen RNeasy plant mini kit (Qiagen) and the total RNA was treated in 1× buffer with 2 U of DNAse I (Ambion, Austin, TX, USA) added to the reaction and incubated for 30 min at 37°C.
For deglycosylation reactions, samples denatured in protein loading buffer were treated with 500 units of PNGase F (NEB) in 1× G7 Reaction Buffer and 1% NP40 for 1.5 hours at 37°C.
Each tube corresponding to a time point was halved and one half was treated in 4X Laemmli buffer containing 3% β-mercaptoethanol and the other half in 4X buffer without β-mercaptoethanol.
The next day, the tail tips in lysis buffer were treated and purified with phenol, phenol chloroform isoamyl alcohol, and chloroform.
Controls in which the primary antibody was replaced with buffer were treated identically.
The cells in arginine-containing buffer, arginine-free buffer, and rADI pretreated arginine-containing buffer were treated with NMDA for 1 h.
