All buffers and purification chemicals were of analytical grade.
Pooled frozen tissue from five plants were ground to a fine powder in liquid nitrogen and approximately 100 mg of homogenized tissue was used for total RNA isolation according to manufacturer's protocol, except the homogenized seed tissue was initially extracted in 600 μl of RLT buffer and during purification, samples were incubated in buffer RW1 for 5 min during the column washing step.
The two differentially labeled reactions to be compared were mixed and buffer exchange, purification, and concentration was accomplished by microcon-10 (Amicon) filtration.
The soluble fraction was kept for purification via nickel-nitriloacetic acid beads using 50 mM Tris (pH 8), 0.3 M sodium chloride, and 0.3 M imidazole as the elution buffer, and an extra purification step through size-exclusion chromatography using a HiLoad 16/60 Superdex 75 prep-grade column (GE Healthcare) gave pure protein.
Briefly, lysates were incubated with HaloLink™ resin, followed by washing with HaloTag® Protein Purification buffer and cleavage with TEV Protease Cleavage Solution (HaloTag® Protein Purification buffer supplemented with 1/16 volume TEV protease) on a rotator (NeoLab, Heidelberg, Germany) for 1 h at RT.
In experiments where Gas6 control is designated TBS, purified Gas6 was used for Gas6 stimulation (Gas6), and purification buffer as control (TBS).
The aqueous phase was transferred into a new tube and added to 30 µl of Direct Purification Buffer and 25 µl of Wizard PCR Preps DNA purification resin (Promega) per oocyte.
Our results support recommendations that effective In vitro suppression of endogenous protein-phosphatase activity necessitates the inclusion of a wide assortment of protein-phosphatase inhibitors (e.g. NaF, MoO4−, VO3−, PPi and microcystin-LR) in extraction and purification buffers [ 34].
Selenomethionyl-derivatived crystals were grown the same way as the native protein crystals, except the culture media used was defined media with selenomethionine additive (Molecular Dimensions, Altamonte Springs, FL) and purification buffers after the last Ni2+ column elution contained 10 mM DTT (instead of β-mercaptoethanol) and 0.5 mM EDTA.
The buffer throughout purification and subsequent procedures was 0.1 M Tris-HCl, pH 7.6, with 5 mM EDTA.
