Bio spin columns p 30

Equilibrium peptide binding to TmrAB was examined using scintillation proximity assays (SPA). TmrAB (0.2 µM) reconstituted in lipid nanodiscs was mixed with the peptide RRYQKSTEL (9.75 µM of R9LQK) traced with tritylated RRY-[³H]L-KSTEL (0.25 µM of ³H-R9L, Hartmann Analytic). To induce the IF-to-OF transition, TmrA^EQB was incubated with 1 mM ATP (Sigma-Aldrich) and 3 mM MgCl₂ for 5 min at 45 °C. If indicated, the excess of free ATP and peptide was removed by rapid gel filtration (Bio-Spin columns P-30, Bio-Rad). Copper-chelated SPA beads (PerkinElmer) were added to a final concentration of 5 mg/ml. The scintillation proximity assay was performed at 20 °C in cpm mode (Wallac MicroBeta). Background was determined in the presence of 200 mM imidazole. Data represent mean ± SD from three experiments. The total binding signal was background-corrected. The specific binding was multiplied by the dilution factor of radiolabeled ³H-R9L. The recovery of peptide binding during the OF-to-IF transition was followed by mono-exponential fit $Y=Y_0+(Plateau-Y_0)\bullet (1-e^{kt})$ with Y: binding, Y₀: binding at X = 0, k: rate constant. The half-life was calculated as $t_{1/2}=\frac{ln2}{k}$
Peptide binding was analyzed by a one-site Langmuir-type binding model $Y=\frac{B_{max}\bullet \left[P\right]}{K_D+\left[P\right]}$ with Y: binding, B_max: maximum binding, and K_D: equilibrium dissociation constant.

The plasmids were linearized with NotI restriction endonuclease (NEB) and transcribed using the mMessage mMachine SP6 Kit (Ambion) to produce capped RNA. Synthetic RNA was purified with P-30 Bio-Spin columns (Bio-Rad, Hercules, CA) followed by phenol/chloroform extraction and ethanol precipitation, and RNA concentration was quantified by Nanodrop (Thermo Fisher Scientific, Waltham, MA) and estimation of agarose gel electrophoresis bands.

Excised PCR products were purified using the Ultrafree-DA Centrifugal purification system (Millipore/Amicon, Bellerica, MA, USA) and sequenced by the Idaho State University Molecular Research Core Facility (Pocatello, ID, USA). Purified products were ligated into the PCRII vector (Invitrogen, Carlsbad, CA, USA) overnight at 14 °C. The ligation product was transformed into chemically competent TOP10 Escherichia coli cells (Invitrogen, Carlsbad, CA, USA). Sequence analysis was performed by the Idaho State University Molecular Research Core Facility (Pocatello, ID, USA).
Five micrograms of riboprobe-containing plasmids were linearized using 5U Hind III with 10× BSA in 20 µL total volume. Plasmid digest fragments were subsequently purified by phenol/chloroform extraction. One-tenth volume of 8 M LiCl was added to each reaction followed by the addition of 2.5 volumes of 100% ethanol. One microgram of linearized/purified plasmid was used as a template to synthesize antisense, digoxigenin-labeled probe using DIG RNA Labeling (Roche Applied Science, Indianapolis, IN, USA). A control probe was synthesized using pSPT18-neo empty plasmid. Riboprobe synthesis products were purified using P-30 Bio Spin columns (BioRad Laboratories, Hercules, CA, USA). Probe reactions were then diluted to a working concentration with hybridization buffer for in situ hybridization assays.