Spin tagging methods

Quantitation of the CBF can be an important factor in studies of therapeutic intervention. The method of arterial spin-labeled (ASF) perfusion MRI allows quantitative determination of the perfusion rate in units of ml/100 g tissue/min. However, the technique requires exceptional instrumental stability, as the result depends on accurate determination of the difference between tw o images on the order of a few percent. With the appropriate acquisition parameters to achieve sufficient signal to noise ratio (>100) and current instrument stability, this technique is feasible, especially in high field clinical systems. Currently, arterial spin-tagged perfusion measures are not widely used in clinical settings. 

Arterial spin-tagged perfusion MRI is performed by inverting the v ater protons in the blood of the arteries supplying blood to the tissue of interest. Typically, in brain, the carotid arteries are used as the supply. The original description of the method (Detre et al., 1992 Williams et al., 1992) uses a continuous narrow band RF inversion of the protons in the arterial blood, outside of the imaged slice, before and after excitation to cause a steady-state loss of signal in cerebral tissue. The signal loss is related to tissue perfusion (Detre et al., 1992) by ... 

Leave to express for 3-4 days, then collect medium, spin, filter 0.22 pm, then go on to protein purification (method depends on the tags used etc.). 

Figure 1.4 Results from the affinity purification method. (A) Purification of a 94-nt RNA from a 100-gl transcription reaction, using a QIAGEN Ni-NTA spin column. Lane Tx the raw transcription lane FT column flow-through lanes Wl — W3 column washes lanes El and E2 column elutions lane s imidazole regeneration. Bands a , b , and c indicate the full-length transcript,. V-tag, and product RNA, respectively. (B) Purification of the same RNA as panel (A), but from a larger-scale (3.25 ml) reaction using 3 ml of Ni-NTA resin in a gravity flow column. Labeled identically to (A), but with a third elution step. Figure is reprinted with permission from Batey and Kieft (2007).

The CD-tagging database uses internal GFP fusions rather than terminal fusions (Jarvik et al. 1996), as discussed above. The GFP-tagged cells were imaged using a laser spinning disk confocal microscope at 60x. Clustering methods were used to analyze the location patterns of the tagged proteins. 

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