Spin columns

The in vitro transcribed RNAs are phenol-chloroform extracted, ethanol-precipitated, and washed once with 70% ethanol. The RNA pellet is resuspended in 30 1 H20 and loaded to DyeEx 2.0 spin columns (Qiagen) to remove free nucleotides the RNA is then quantified by spectrophotometry. Approximately 20 to 30 pg of RNA is obtained from one reaction. For the initial preparation of transcripts, we would examine the quality and quantity of synthesized RNA by separating them on 1% denaturing agarose gels with total cell RNA preparation as a size maker (28S and 18S ribosomal RNAs... 

Following linearization of plasmids (Fig. 13.5), the DNA is phenol chloroform extracted, passed through a Sephadex G50 spin column, and precipitated with 2.5 volume of ethanol and hjth volume of 3MNaOAc (pH 5.2). The pelleted DNA is washed with 70% ethanol, dried, and resuspended in water at a final concentration of 1 pg/pi and stored at —20°. 

Oxidation of the cap structure To 400 fd of 32P-cap-labeled mRNA, 1.6 fd of 100 mMNaI04 is added and left on ice for 2 h, protected from light. The reaction is terminated by addition of 20 fd of 50% glycerol, passed through a G50 spin column and ethanol precipitated. The pelleted... 

Remove excess reagent and reaction by-products by dialysis or gel filtration using 0.1M sodium phosphate, 0.15M NaCl, lOmM EDTA, pH 7.5. For chromatographic separation, use a desalting gel filtration support such as the Zeba desalting spin columns (Thermo Fisher) or the equivalent. The SAMSA-modified protein may be stored at -20°C until needed. 

Separate excess cystamine and EDC (and reaction by-products) from the modified protein by dialysis or gel filtration using 10 mM sodium phosphate, 0.15M NaCl, pH 7.2. A desalting column may be used for the gel filtration procedure (i.e., Zeba spin columns from Thermo Fisher). 

Purify the modified protein from reaction by-products by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. Alternatively, centrifugal spin columns containing a desalting resin may be used for rapid purification (Thermo Fisher). 

Immediately purify the maleimide-activated (strept)avidin away from excess crosslinker and reaction by-products by gel filtration on a desalting resin. A spin column will facilitate the... 

G-50 eluted in saline (150 mM NaCl), as described above. Alternatively, the gradient could be formed using spin columns (spin 2 x 100 pL), and pooling the fractions. 

Ciprofloxacin (Bayer Corporation) is often loaded at a D/L ratio of 0.3mol mol. After preparation of a ciprofloxacin standard solution (4mM in water), 5 pmol of lipid and 1.5 pmol of ciprofloxacin are pipetted into a glass test tube (or plastic Ependorf tube), adding saline to give a final volume of 1 mL (5 mM lipid concentration). This solution is incubated at 65°C for 30 minutes, with aliquots (50-100 pL) withdrawn at appropriate time points (0, 5, 15, and 30 minutes) and applied to a spin column [centrifuge at 2000 rpm (x670g) for two minutes]. An aliquot (50 pL) of the initial lipid-drug mixture is saved for determination of initial D/L. 

The columns are here designed specifically for a defined purpose. These columns are easier to use, faster and they require much less resources. Some of the columns include the NAP-25 or PD 10 desalting columns (from Amersham Biosciences), His Tag columns such as Ni-NTA spin column from Qiagen, His Bind from Novagen or His GraviTrap from GE Healthcare. 

A spin column from the kit is inserted into a 2-mL collection tube, and the sample is pipeted onto the column. The column is centrifuged at 1 l,000g for 1 min, and the flowthrough is saved to evaluate the cDNA synthesis efficiency. The collection tube is discarded in an appropriate container for radioactive waste. 

The spin column is now washed three times by inserting the column into a fresh 2-mL collection tube, adding 400 pL buffer NT3 to the column, and centrifuging as described in step 9. Both the collection tube and the flowthrough are discarded. 

To elute the cDNA synthesis probe, the spin column is inserted into a clean 1.5-mL microcentrifuge mbe 100 pL buffer NE are added to the column and soaked into it for 2 min. The column is centrifuged as described in step 9, with the flowthrough now containing the labeled cDNA probe. 

DNA was isolated from 5 x 10 spleen cells using QIAamp spin columns (QIAGEN Blood Mini Kit, Hilden, Germany) according to the manufacturer s instructions. DNA concentrations were determined on a spectrophotometer. 

Drug Screening Using Gel Permeation Chromatography Spin Columns Coupled with ESI-MS... 

A number of indirect methods have been developed with mass spectrometric detection to rapidly study non-covalent complexes for drug screening purposes [2]. Among the most promising and simple indirect methods that overcome the limitations described above for directly studying non-covalent complexes by mass spectrometry is the application of size exclusion techniques in the spin column format for the screening and analysis of drug-protein complexes under optimum mass spectral sensitivity conditions [11-13]. 

Fig. 2.1 CPC spin column used for isolating protein/RNA-drug non-covalent complexes in the eluate upon centrifugation and the ESI-MS steps to detect the ligands upon denaturing of the protein/RNA-drug non-covalent complexes. (A) Spin column cartoon, (B) Photo of a miniature CPC spin column, (C) Schematic of CPC spin column/ESI-MS procedures.

B) CMVP C87/138/161A/A144D + DFMK (impure). After Spin Column... 

Fig. 2.2 ESI mass spectra obtained from the CPC spin column/ESI-MS screening assay of non-covalently bound protease-inhibitor complexes. Enzymatically active CMVP A144D/C87A/C138A/C161A A/as used in this experiment. (A) Reference ESI mass spectrum of impure inhibitor DFMK (MW 988.5 Da). (B) ESI mass spectrum of the spin column eluate of CMVP A144D/C87A/ C138A/C161A and DFMK, incubated at a...

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