Metabolite extraction and quenching were performed on plate with 1.5 mL ice cold extraction medium (90% methanol, 10% water) containing 1 µg/mL ribitol (A5502, Sigma Aldrich), phenyl β-d -glucopyranoside (292710, Sigma Aldrich), isoguanosine (sc-207768, Santa Cruz, Dallas, TX, USA), d4-succinate (293075, Sigma Aldrich) and methyl-tyrosine (M8131, Sigma Aldrich) as the internal standard. Cells were detached on ice by using a cell scraper, transferred into screw-cap tubes prefilled with 300 mg glass beads (G4649, Sigma Aldrich) and immediately frozen in liquid nitrogen until homogenization. Cells were homogenized using a Precellys tissue homogenizer (P000669-PR240-A, Bertin instruments, Montigny-le-Bretonneux, France) at −10 °C. Three cycles of homogenization for 15 s at 6500 rpm were applied with 10-s breaks in between cycles. Samples were then centrifuged (20,000× g) for 10 min at 4 °C to remove cell debris and protein precipitates, and 500 µL of each supernatant was transferred into two new reaction tubes for GC-MS and LC-MS analyses. Finally, extracts were dried using a vacuum rotator (Eppendorf, Hamburg, Germany) and flushed with nitrogen. The DNA content in the extracts was measured using NanoDrop 1000 (Thermo Fisher Scientific).
G4649
Manufactured by Merck Group
G4649 is a laboratory equipment product manufactured by Merck Group. It is a device designed for specific laboratory functions. The core function of this product is to enable researchers and scientists to perform various tasks and experiments within a controlled laboratory environment. No further details or interpretation about its intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
A5502, by Merck Group (1 mentions)
Phenyl β d glucopyranoside, by Merck Group (1 mentions)
D4 succinate, by Merck Group (1 mentions)
Vacuum rotator, by Eppendorf (1 mentions)
Precellys tissue homogenizer, by Bertin Technologies (1 mentions)
Glass beads, by Thermo Fisher Scientific (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Penicillin g, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
P35g 1.5 14 c, by MatTek (1 mentions)
G1277, by Merck Group (1 mentions)
2 d quant kit, by GE Healthcare (1 mentions)
5 protocols using g4649
1
Metabolite Extraction and Quantification
2
Bead-Loaded Fluorescent Fab Delivery
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Cells were cultured in glass-bottom dishes (35 mm, 14 mm glass, Mat-Tek). The next day dye-conjugated Fabs were loaded into the cells through bead-loading22 (link),27 (link),29 (link),66 (link),67 (link), as follows: First, the fluorescent Fabs (CTD-RNAP2-CF640 and Ser5ph-RNAP2-Cy3, ~1 mg/mL, each) were mixed with PBS up to 4 μL in the cell culture hood. Second, the medium was removed completely from the dish and stored, and the Fab mixture was added to the center of the dish. Third, glass beads (106 μm, Sigma-Aldrich, G-4649) were immediately sprinkled on top before cells dried up and the dish was tapped ~10 times against the bench. This tapping causes the beads to roll over cells and induce small tears into which the Fab can diffuse in. Fourth, the stored medium was quickly added back to the cells, again to prevent cells from drying out. Cells were then placed in the incubator to recover for 1–2 h. Post-recovery, the glass beads were gently washed out with phenol red-free DMEM (DMEM−, Thermo Fisher Scientific, 31053-028), and the cells were stored in DMEM+ medium (DMEM− supplemented with 10% FBS, 10 U/mL P/S, and 1 mM l -glut) for live-imaging experiments.
3
Protein Extraction from Yeast Cells
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Protein extracts were prepared as follows: Cells were washed with ice-cold PBS and collected into a centrifuge tube in either glass beads lysis buffer (GBL: 50 mM Tris, pH 7.5, 5 mM MgCl2, 0.5 mM EDTA, and 250 mM Sucrose) or glass beads lysis buffer plus 0.2% NP-40 (GBLN). One volume of acid-washed glass beads (Sigma Aldrich, G4649) per 2–3 volumes of ice-cold glass bead buffer (+1 mM DTT) was added to the tube containing the cells and buffer followed by vortexing (10 times in 30 s bursts, returning the samples to the ice for 1–2 min in between) and centrifugation (14,000 g, 4°C, 25 min) in order to pellet the glass beads, nuclei, and membranes. The supernatant was carefully transferred to fresh Eppendorf tubes and the pellets were discarded. The protein concentration was determined via the Thermo BCA protein assay kit. For Western blotting, 25 μg of protein was then fractionated on Tris–glycine SDS-PAGE gradient (4–15% acrylamide) gels, transferred onto PVDF membranes, and detected with the indicated antibodies using a LI-COR detection system.
4
RNA Bead-Loading for Live-Cell Imaging
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HeLa cells were sub-cultured into a glass bottom dish (P35G-1.5-14-C; MatTek, Ashland, MA) using DMEM/F12 (Thermo Fisher) supplemented with 10% fetal bovine serum (Biowest, VWR #S1810-500), 100 U/ml penicillin G (Sigma-Aldrich, St. Louis, MO), and 100 μg/ml streptomycin (Sigma-Aldrich), at 37°C and 5% CO2, 1 day prior to RNA-loading. AF647-labeled RNAs in water were loaded using a bead-loading method (46 (link),47 (link)) as follows: The culturing medium was removed and replaced with a solution containing 2 μM of either AF647-labeled RNA or of the dye itself, together with glass-beads (<106 μm; G4649, Sigma-Aldrich). The glass bottom dish was then gently tapped against the workbench 10 times, after which the glass-bead and RNA solution was removed by washing in PBS three times, before finally adding Opti-MEM I (Thermo Fisher). Measurements were started within minutes after loading and were finished within 2 hours.
5
Protein Extraction from A. brasilense
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A. brasilense strains were grown in NB media at 30°C with shaking and bacteria in logarithmic phase (A600 0.5–0.8) were collected by centrifugation. For flocculation conditions, bacteria were collected by centrifugation after 3–4 hours shaking at 30°C in flocculation medium [6] (link). About 50 mg cells were lysed and homogenized using a mini beadbeater containing a mixture of glass beads (G4649 and G1277, Sigma) and lysis buffer (30% sucrose, 0.1 M Tris pH 8.0, 2 mM PMSF, 1% DTT, 100 mM KCl, 5 mM EDTA) 4 times for 30 sec. Protein extraction was performed from the lysates by phenol extraction [16] (link) and proteins were precipitated overnight using 5 volumes of 0.1 M ammonium acetate in methanol at −20°C. The protein solution was subsequently centrifuged at 6,000 g for 10 min and the pellet rinsed twice with 0.1 M ammonium acetate in methanol, three times with cold methanol and once with 80% acetone. The protein pellet was then cold-dried using a vacuum freeze dryer for 4 hours, then dissolved in IEF buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 0.5% IPG buffer pH 3–10, 1% DTT, 0.2% Coomassie Brilliant Blue). The protein concentration was determined using the 2D Quant Kit (GE Healthcare Life Science, Australia).
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A. brasilense strains were grown in NB media at 30°C with shaking and bacteria in logarithmic phase (A600 0.5–0.8) were collected by centrifugation. For flocculation conditions, bacteria were collected by centrifugation after 3–4 hours shaking at 30°C in flocculation medium [6] (link). About 50 mg cells were lysed and homogenized using a mini beadbeater containing a mixture of glass beads (G4649 and G1277, Sigma) and lysis buffer (30% sucrose, 0.1 M Tris pH 8.0, 2 mM PMSF, 1% DTT, 100 mM KCl, 5 mM EDTA) 4 times for 30 sec. Protein extraction was performed from the lysates by phenol extraction [16] (link) and proteins were precipitated overnight using 5 volumes of 0.1 M ammonium acetate in methanol at −20°C. The protein solution was subsequently centrifuged at 6,000 g for 10 min and the pellet rinsed twice with 0.1 M ammonium acetate in methanol, three times with cold methanol and once with 80% acetone. The protein pellet was then cold-dried using a vacuum freeze dryer for 4 hours, then dissolved in IEF buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 0.5% IPG buffer pH 3–10, 1% DTT, 0.2% Coomassie Brilliant Blue). The protein concentration was determined using the 2D Quant Kit (GE Healthcare Life Science, Australia).
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