FAQ of DARTS experiment.docx

此文档收集于网络，如有侵权，请联系网站删除1. How should I choose a cell line? Generally speaking, commonly used cells such as HEK 293, Jurkat, HeLa, MEFs, and even yeast can be (and have been) used. However, your favorite system should be used if the biological effect of your small molecule is, or is suspected to be, specific to a particular cell type, or cell state, e.g., UV-irradiated cells, differentiated cells, etc. DARTS is applicable to any cell, tissue and organism. 2. What lysis buffer should I use? Cell lysates can be prepared using M-PER (Pierce) or a standard Triton X-100 lysis buffer 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM DTT, supplemented with protease and phosphatase (or other, if necessary) inhibitor cocktails. Buffer conditions are not very important for DARTS in our experience; only very denaturing conditions like the RIPA buffer would significantly decrease DARTS efficiency. 3. Why add protease inhibitor cocktail to the lysis buffer? (Will the protease inhibitors affect the proteolytic activity of thermolysin/subtilisin?) Protease and phosphatase (or other) inhibitor cocktails (Roche, Pierce) are included in the lysis buffer at standard concentrations to prevent premature degradation and other alterations to cellular proteins (potential targets). These inhibitors will not significantly affect exogenous proteases (added in excess) later in the experiment (including thermolysin, subtilisin and Pronase). 4. How much protein should I use? What is the protein concentration for proteolysis? We normally use 50 g protein from a fresh cell lysate in a total of 10 L DARTS reaction (can be scaled up or down as necessary*). For example, 5 g/L protein can be incubated with each small molecule, as well as a vehicle control, for 15-30 min at 37 (or alternatively room temperature or 4 ) to allow binding equilibria to establish, followed by DARTS using Pronase (10 mg/mL stock) at 1:300, 1:1000, and 1:3000 protease:protein ratios for 30 min. Our experience has found that nearly all proteins can be partially or completely digested within this range of Pronase concentrations, and therefore nearly all protein targets should exhibit differential digestion in at least one of these conditions. Proteolysis is stopped by adding excess protease inhibitor solution and transferring the samples to ice. Samples will then be analyzed using quantitative proteomics to identify binding targets of each small molecule (more info can be found here). * DARTS scale up:The (50 g in 10 L) can be scaled up as necessary, depending on which proteomics detection method will be used. For example, 50 g is plenty for 1D SDS-PAGE, Western blotting, and MudPIT, but some other proteomics techniques would require more. 5. What concentration of compound should I use? For low (or unknown) affinity ligands, we recommend incubating fresh lysates ( 5 g/L protein) with up to 500 M of compound. The high concentration of compound will help ensure even weak binders can be identified; from our experience we have not seen non-specific protective effects towards non-target proteins in DARTS even at 1 mM concentration or higher of small molecules.For high affinity ligands, low nM concentrations of compound have worked (see Fig. S3), e.g., 2 nM celecoxib (affinity for COX-2: Kd = 2.3 nM) and 10 nM rapamycin; lower concentrations may still work but have not been tested. In any case, we cannot overemphasize the importance of having proper controls - in order to ascertain that stabilization of the putative protein target is specifically caused by ligand binding. For example, unchanged protease sensitivity of an internal control protein(s) which is proteolysed equally across would rule out the possibility that the observed target stabilization might instead be due to fortuitous inhibition of the DARTS protease(s) by the compound. 6. How much protease should I use? For 50 g protein (cell lysate) in a total of 10 L DARTS reaction, use Pronase (10 mg/mL stock) at 1:300, 1:1000, and 1:3000 protease:protein ratios for 30 min. Our experience has found that nearly all proteins can be partially or completely digested within this range of Pronase concentrations, and therefore nearly all protein targets should exhibit differential digestion in at least one of these conditions. Proteolysis is stopped by adding excess protease inhibitor solution and transferring the samples to ice.If necessary, thermolysin (2 mg/mL stock) also works well for many proteins. The initial recommended thermolysin:protein ratios would be 1:10, 1:100, and 1:1000. 7. Should DARTS be performed with compound-treated cells or compound added directly to lysates? In most cases we add our compounds directly to the protein lysates of untreated cells. We generally prefer this in vitro DARTS approach in order to prevent any drug-induced changes in protein levels (expression or turnover), post-translational modifications (such as those that may signal to the proteasome, aggresome, or caspases and other endogenous proteases), etc., that may occur during the cellular response to the drug. Such changes would not be solely caused by drug binding to its target protein and may confound the DARTS assay. (On the other hand, identification of such changes may still provide valuable information about the drugs cellular mechanism of action.)But at least in the cases of didemnin B, rapamycin, and SMER3, the cells could also be treated with the compounds (without adding the compounds again during cell lysis or DARTS proteolysis) and protection of the target proteins was just as good as when the compounds were added directly to cell lysates (of untreated cells). For some weaker binding compounds, however, it may be worthwhile adding the drug both to the cells and again into the lysate to help ensure saturation binding of the target proteins. Another potential advantage of in vivo compound treatment would be to identify target proteins of drug metabolites. Some small molecules may require chemical modifications inside the cell for their biological activity, and such metabolism of the parent molecules may not occur in the cell lysates. 8. What are good control proteins for DARTS? In theory, any non-target protein can serve as a control in DARTS, e.g., an unrelated protein, or a non-binding mutant (if available), etc. That said, however, it is handy to be able to use some commonly used control proteins for which good antibodies are readily available for Western blotting. Although GAPDH generally serves as a good loading control, it may not be the right DARTS control in every case since GAPDH is relatively resistant to proteases. For example, GAPDH is completely resistant to thermolysin; -actin and -tubulin are sensitive to thermolysin and can serve as DARTS controls (except when the control itself is targeted by the small molecule of interest, of course). With pronase, there is good digestion of GAPDH at 1:1000 and especially at 1:300, but there is very little or no GAPDH digestion below 1:1000. (For some target proteins, 1:300 will be over-proteolysing and thus even ligand-bound target proteins are no longer protected.) -tubulin is useful for pronase at 1:1000 to 1:5000, and -actin can be useful for pronase from 1:5000 to 1:10,000.EF-1A could also be a control in a similar range as -tubulin, if necessary. 9. (On pg. 21988) after centrifugation, lysates were diluted in M-PER or reaction buffer? How much dilution is needed? Lysates are to be dil