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MB Sample ID: SA249786
| Local Sample ID: | MED1 Library 100 nM Input Sample 1 |
| Subject ID: | SU002593 |
| Subject Type: | Synthetic sample |
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Collection:
| Collection ID: | CO002586 |
| Collection Summary: | Chemical library to perform condensate metabolomics with a defined set of lipids. To analyze a defined set of lipids at known concentrations, the following molecules were purchased: phosphatidylcholine (3:0/3:0)(Cayman Chemical, 32703), phosphatidylcholine (9:0/9:0)(Cayman Chemical, 10009874), phosphatidylcholine (12:0/12:0)(Echelon Biosciences, L-1112), phosphatidylcholine (16:0/16:0)(Echelon Biosciences, L-1116), phosphatidylcholine (18:0/18:0)(Echelon Biosciences, L-1118), sn-glycero-3-phosphocholine, lysophosphatidylcholine (16:0)(Echelon Biosciences, L-1516), palmitic acid (Sigma Aldrich, P5585), phosphatidylethanolamine (16:0/16:0)(Avanti Polar Lipids, 850705), phosphatidylglycerol (16:0/16:0)(Avanti Polar Lipids, 840455), phosphatidylinositol (16:0/16:0)(Echelon Biosciences, P-0016), PIP2 (16:0/16:0)(Echelon Biosciences, P-4516), phosphatidylserine (16:0/16:0)(Echelon Biosciences, L-3116). These molecules were first dissolved in an appropriate organic solvent and then either 0.33 pmoles, 3.3 pmoles, or 33 pmoles of each molecule were combined in an eppendorf tube. The organic solvents were removed using a SpeedVac Concentrator (Savant, SPD131DDA) at 25oC and the dried chemical libraries were stored at -80oC. Each tube containing a chemical library was used to perform a single condensate metabolomics experiment. These libraries were initially re-suspended in condensate buffer (50 mM NH4HCO3 pH 7.5, 50 mM NaCl, 1 mM DTT). Molecules that were not fully soluble in condensate buffer were removed by centrifugation (2x5 min, 16,000 g each), in which only the supernatant was retained. Due to the lack of crowding agents, phase separation required greater concentrations of protein and RNA than typically employed for nucleocapsid and MED1 condensate formation17,32. Purified protein (37.5 μM) was centrifuged (1 min, 1,000 g) to disrupt any existing condensates and to remove any precipitated proteins. Purified protein (final concentration, 30 μM) was combined with metabolites (final concentration, 150 g/l protein equivalent) and then phage lambda RNA (final concentration, 0.15 μM) in a total volume of 300 µl. An input sample (10 µl) was saved and then the sample was allowed to incubate for 10 min at 25oC. Condensates were then separated from the aqueous environment by centrifugation (10 min, 12,500 g, 25oC). The aqueous phase was removed from the condensate phase and then equal volumes (usually ~ 2 µl) of the aqueous fraction, condensate fraction and input sample were processed for metabolomics using identical approaches as described below. First the samples were diluted in ammonium bicarbonate buffer (50 mM NH4HCO3 pH 7.5) and briefly heated (2 min, 65oC) to disrupt condensates before being added immediately to 4x volume of ice-cold 100% methanol to precipitate protein and RNA. Protein and RNA were separated from metabolites by vortexing the samples (2 min), followed by incubation at -25oC (10 min) and then centrifugation (5 min, 13,000 rpm). The supernatant was saved and the process was repeated on the pellet two more times after adding 200 µl of 80% methanol each time to the pellet. The three supernatants were combined and centrifuged (10 min, 14000 rpm) to remove any additional macromolecules. The final supernatant was collected and dried using a SpeedVac Concentrator run at 25oC. |
| Sample Type: | Synthetic Mixture |
| Collection Method: | 80% methanol |
| Storage Conditions: | -80℃ |
