Fretting test methods

However, FLIM measurements by both frequency and time domain using commercially available software suffers from variation in the measured lifetime from region to region in cells [36], which can be confusing. In the light of the potential pitfalls associated with each of the above-mentioned FRET techniques, potential protein associations should ideally be tested independently by a combination of two or more FRET-based methods in addition to biochemical techniques such as co-immunoprecipitations. 

H. Helmi Attia, R.B. Waterhouse Standardization of Fretting Fatigue Test Methods... 

ASTM International has a number of standard test methods for erosion, cavitation, and fretting wear. 

A number of test methods are used to evaluate the fretting resistance of various materials and lubricants. The object of the tests is to bring two surfaces into contact under a known load and contact geometry and to move one surface relative to the other under an oscillating motion with a very small amplitude. 

The above test methods do not exactly simulate the actual fretting conditions. The surface damage produced by the test should be compared to the damage on parts damaged in service. If there is significant difference between scar morphologies, the wear mode may not be correctly simulated in the test. Corrections can be made in contact geometry, contact stress, and amplitude and velocities of movement. 

ASTM D 4170-97 Standard Test Method for Fretting Wear Protection by Lubricating Greases, Annual Book of ASTM Standards, Vol. 05.02, ASTM International, West Conshohocken, PA. 

TABLE 10—Test method for evaluating fretting corrosion. 

Standard Test Method for Measuring Fretting Corrosion of Osteosynthesis Plates and Screws Standard Test Method for Cavitation Erosion Using Vibratory Apparatus Standard Terminology Relating to Wear and Erosion... 

Fretting wear D4170 Test Method for Fretting Wear Protection al Lulnicating Greases Mass 1< ratio... 

Another important lesson from Table 8.1 is that all three of the methods tested yielded virtually the same FRET efficiencies for the same samples. The sRET method as implemented used two-photon excitation on a Zeiss 510 META/NLO microscope, as did the FLIM-FRET method, but FLIM-FRET used auxiliary time... 

Selected entries from Methods in Enzymology [vol, page(s)] Analysis of GTP-binding/GTPase cycle of G protein, 237, 411-412 applications, 240, 216-217, 247 246, 301-302 [diffusion rates, 246, 303 distance of closest approach, 246, 303 DNA (Holliday junctions, 246, 325-326 hybridization, 246, 324 structure, 246, 322-324) dye development, 246, 303, 328 reaction kinetics, 246, 18, 302-303, 322] computer programs for testing, 240, 243-247 conformational distribution determination, 240, 247-253 decay evaluation [donor fluorescence decay, 240, 230-234, 249-250, 252 exponential approximation of exact theoretical decay, 240, 222-229 linked systems, 240, 234-237, 249-253 randomly distributed fluorophores, 240, 237-243] diffusion coefficient determination, 240, 248, 250-251 diffusion-enhanced FRET, 246, 326-328 distance measurement [accuracy, 246, 330 effect of dye orientation, 246, 305, 312-313 limitations, 246,... 

FRET-based readouts are the most common methods applied for endopeptidase activity assays. In general, the FRET readout is simple, numerous assay examples are published, many fluorescently labeled substrates are commercially available, and labeling procedures are well established. Furthermore, broad range of fluorescence donors and acceptors is available. FRET quench substrates allow fast and cost-efficient operations in an industrialized HTS environment. However, in many cases, the assays suffer from fluorescence artifacts from the tested compounds. This can lead to falsepositive and false-negative results because the most frequently used FRET quench dyes are excited in the ultraviolet range. 

In another study, 42,000 compounds were screened in three different versions of a farnesoid X receptor (FXR) antagonism assay. AlphaScreen, time resolved fluorescence (TRF), and time resolved FRET (TR-FRET) were used. These identified 104, 23, and 57 actives, respectively. Only 18 compounds were identified by all three methods. Further cellular testing showed that only about 30% of them were functionally active, with AlphaScreen identifying more of the functionally active compounds than the other two methods. This group also found that screening mixtures rather than discrete compounds was more likely to result in both false positives and false negatives. ... 

Several methods are available today to test candidate ion channel active drugs (ICADs) electrophysiology (patch clamp), binding assays, radioactive flux assays, membrane potential-sensitive fluorescent dyes, ion-sensitive dyes, and voltage sensing based on fluorescence resonant energy transfer (FRET). 

The plotted characteristic numbers of cycles to fracture were determined by the Weibull method [21]. Each point represents a test series of ten specimens. In the diagram it can be seen clearly that fretting fatigue leads to a distinct deterioration of the specimen strength and life time. The higher the maximum Hertzian stresses the clearer the decline of the strength and life time is. 

In Figure 10 the number of fracture cycles and the characteristic number of fracture cycles for the respective test series is plotted against the maximum principal stress on the tensile loaded side of the specimens. The characteristic number of fracture cycles was determined by the method developed by Weibull [21]. In the Woehler diagram it can be seen clearly that fretting fatigue leads to a distinct deterioration of the life time. The higher the maximum stresses the clearer the decline of life time is. At Fn = 10 N (Pmax = 2311 MPa) the life time decreases at a maximum base load of OR.max = 210 MPa for about 80%. at Fn = 20 N (pmtx -2912 MPa) the life time decreases for about 91% compared to to life time under the same maximum base loading. 

---