X-Filter

Finally, the use of side-stream filters is recommended. Bag filters generally are satisfactory, although for the removal of the finest iron particles, an in-series twin-bag system may be required use 5 to lOjx bags followed by 1 to 2 x. Where only one bag filter is employed, use either conventional 5 x filters or high-quality depth filters. 

Filter housings fitted with (provide filter pore size) X filter cartridges (product specific)... 

Syringe (5 mL) with 0.8 /x filter system Constant-temperature water bath, 60°C... 

The NOE matching protocol is described pictorially in Figure 5.1. Two files are needed for input an experimental NOE peak list with ligand protons assigned and a set of trial binding poses to be evaluated or scored. The list of experimental peaks is typically derived from a 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectrum.1116-201 (Hereafter, this type of spectrum will be referred to as a 3D X-filtered NOESY spectrum.)... 

Figure 5.6 (A) COST versus the RMSD (A) to the target pose for CDK2-/4. The predicted protein chemical shifts were set to the corresponding BMRB average values. The 3D X-filtered NOESY spectrum used as input for NOE matching was simulated from the target structure. (B) Superposition of target pose and the minimum cost pose (dark gray) from (A).

In this section, some of the approaches described above for enhancing the sensitivity and information content of protein-ligand NOEs are demonstrated for relatively large protein-inhibitor complexes. In addition, we demonstrate that a medium-quality 3D X-filtered NOESY spectrum can be obtained for a large protein-inhibitor complex by using a stabilized, uniformly 13C/15N-labeled protein sample in conjunction with an elevated experimental temperature to increase the rotational correlation time of the protein-ligand complex. 

Figure 5,20 Portion of a 3D X-filtered NOESY spectrum of uniformly 13C/15N-labeled, stability-enhanced kinaseX in complex with kinaseX inhibitor 2. The protein and inhibitor concentrations used were 300 pM. The F3 (inhibitor1H) plane is at 7.83 ppm. Peaks with protein resonance assignments are labeled. (Note Val-A and Val-B refer to the y-i and y methyl, respectively of the same valine residue.) The spectrum was recorded at 35 °C, 600 MHz 1H frequency using a NOESY mixing time of 100 ms on a Varian Inova spectrometer equipped with a Cold Probe. The spectrum is aliased in the 13C (F2) dimension.

Methanol (LC grade) filtered through a 0.45-/X filter using a solvent clarification device. 

Prepare an MTT stock solution of 5 mg mH (Sigma, St Louis) in phosphate-buffered saline (PBS), pH 7.5, and filter through a 0.22- x filter to sterilize and remove the small amount of insoluble residue. 

Luy et al. have described a sensitive X-filtered-E.COSY-type method that measures both the sign and magnitude of long-range dipolar couplings... 

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