Filtration assays

The processes of both seed formation and fibril extension are dependent on temperature and on peptide concentration, with 37°C being required for establishing equilibrium within 24 h with 30 pM Pi 4o- A full description of the assay system may be found elsewhere [97,117], A 4 h reaction time is typically within the linear portion of the time course. This nucleus-dependent assay detects mainly inhibitors that are substoichiometric with the monomeric peptide, which is present at high concentration. It is relatively insensitive to inhibitors that target the monomeric peptide. Whether the inhibitors interact with the growing end of a seed or with a low abundance conformational form of the p peptide that is competent to add to the seed is difficult to determine at this time. Similar dose-response curves are obtained for Congo Red as an inhibitor with either thioflavin T (ThT) fluorescence or filtration of radioiodinated peptide readouts (Fig. 4) Caveats in the interpretation of both the ThT and radiometric filtration assays for the evaluation of putative inhibitors are discussed elsewhere [97]. 

D., Melch, M., McConkey, M., Bergeron, J., Wong, S. K.-E. fully automated radioligand binding filtration assay for membrane-bound receptors. BioTechniques 2002, 33, 932-937. 

G Wu, S Daniel-Issakani, K LaMarco, B Strulovici. Automated high throughput filtration assay application to polymerase inhibitor identification. Anal Biochem 245 226-230, 1997. 

Values in units of °C, Values in units of kilodaltons, gel filtration assay, aggregation, no value obtained. 

PPARy and RXRa LBDs exhibited the expected binding to their respective ligands. Novel ligands that had been implicated as PPARa and LXRa effectors in a cell-based assay were found to bind to PPARa and PPAR5 respectively in the gel filtration assay. No ligands were found to bind to LXRa in the gel filtration assay. 

Qualitatively, CD spectral analysis indicated that LXRa was different from the other constructs, all of which exhibited classical a-helical structure. The LXRa spectrum clearly did not contain double minima at 208 and 222 nm but instead had a more narrow trough with a minimum around 220 nm. The spectrum is very similar to that taken from a PPARa sample which we progressed to 60°C and then rescanned (data not shown). This unfolded PPARa did not exhibit ligand binding in the gel filtration assay. Also, LXRa showed heavy scattering below 220 nm. Taken altogether, the CD data suggest that recombinant LXRa as expressed does not share a similar structure with the other four constructs. 

Figure 3-13 Comparison of filtration and scintillation proximity assays, a. In filtration assay, the enzyme, substrate, and inhibitor are mixed the uninhibited enzyme splits the radioactive portion ( ) off the substrate, and filtering the mixture, followed by measuring the radioactivity of the filter, tells how much inhibition has occurred, b. In SPA, the same mixture is treated with resin beads containing a scintillant that fluoresces only in close proximity to the radioactive source Any radioactivity that was split off by the enzyme does not need to be filtered In SPA.

Ten xl of G-protein-containing samples is mixed with 10 [il of preactivated toxin (see section 4.3.1 100 ng PT/tube) and started by addition of another 10 il of reaction buffer (total volume 30fil). The assay is conducted as outlined in section 4.3.3. After termination of the reaction samples may be quantified by filtration assay as described by Sternweis and Pang (1990). 

Dold, K.M. and W.F. Greenlee. Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin... 

Conventional scintillation counters such as the Microbeta (Wallac/Perkin Elmer, Turku, Finland) or the TopCount (Packard, Meriden, USA) use photomultiplier detection systems that count 8 or 12 wells at a time, resulting in a readout time of 40 minutes per 384-well microplate. Bialkali photocathodes (Sb-Rb-Cs or Sb-K-Cs) used in standard photomultipliers have a maximum spectral response at about 420 nm, with a quantum efficiency for detection of up to 30%. Thus, the aforementioned instruments are ideally suited for filtration assays and SPA assays with the blue-emitting YSi and PVT beads. 

G-protein Gi,Gs GTPy35S filtration assay 96 Safety and costs... 

Use of an Eppendorf microfuge to centrifuge cells layered over a sihcone oil of appropriate density permits a very rapid separation of cells from the aqueous suspending medium. The addition of radiolabeled NO3" at 15-s intervals to cells suspended over silicone oil followed by sedimentation established initial rates of transport, and HPLC analysis of methanolic extracts of the pellets identified the transported compound. Using NO3" concentrations from 1-30/xM and incubation times of 0, 15, 30, and 45 s, we established the presence of the high-aflBnity transport system (Table III) obtained earlier (see Table I). The Km of that system when assayed in this manner was about half that obtained by filtration assays. Selected pellets thus obtained were extracted and analyzed by HPLC to determine whether N03 or NO2 was the transported compound (Table IV). Since no N02" or NH4 appear in the pellet in 15-s incubations and no N02" appears after 45 s, we conclude that N03 is the transported compoimd. 

While the goal in this study was to demonstrate the presence of one or more transport system(s) for N03", our initial rate studies were hampered by the uptake, then loss, of counts bound to filters in filtration assays. This suggested that N03 was bound to the cells in some fashion, then released, perhaps as N02", or reduced nitrogen (see also Ref. 15). By collecting the filtrates in these experiments and analyzing them by anion exchange HPLC, we demonstrated that in aerobic cultures NO2 is released into the medium transiently and then is taken back up. Since NO3" may be reduced on the outside of the cell membrane, the transport capability identified in 2.5-min experiments may have reflected the subsequent uptake of NO2" or the assimilation of NO3" after reduction to NH4 ( 6). On the other hand, NO3" may be transported and reduced intracellularly to NO2", which is transiently excreted as a toxic compound... 

Bruns, R., et al. (1983). A Rapid Filtration Assay for Soluble Receptors Using Polyethylenimine-treated Filters, Anal. Biochem. 132 74-81. 

In filtration assays, L is separated from the receptor-ligand complex by rapidly filtering the incubation mixtures through an appropriate support. Most assays call for the use of glass fiber filters. The filters are rapidly rinsed with 5-10 ml of ice-cold assay buffer, placed in counting tubes and radioactivity is quantified as described above. 

NMR Studies In sharp difference to the 24mer, the 28mer can be trapped in pure hairpin conformation after snapcooling in the absence of additional cations, as indicated by gel filtration assays (data not shown). 

Fig. 3. Gel filtration assay of the CCKBP. Protein used in gel filtration assay came from 30% saturated...

Filtration Assay. Nitrocellulose membranes are soaked before use in 25 mAf potassium phosphate (pH 7.4) and 25 vaM MgCL filters which are not wetted within 2 min are discarded. The filter disks are placed on a filter manifold (Hoeffer Scientific) and a gentle vacuum is applied (about 2 ml/min filtration rate) to remove excess moisture. After removal of vacuum, 100-/tl aliquots of the reaction mixture are applied to each disk and allowed to permeate the membrane. The filters are washed at about 2 ml/rain filtration rate with seven 0.5-ml portions of a solution containing 25 mM phosphate buffer (pH 7.4) and 25 vaM MgCl2. The damp filters are dissolved in 10 ml of Bray s solution and counted. 

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