A freshly purified protein sample at 7 mg ml−1 was added to Quantifoil R1.2/1.3 400 mesh Au holey carbon grids (Quantifoil), blotted with Vitrobot Mark IV (FEI), and frozen in liquid ethane. For ML-SA1-bound protein, the protein in a buffer containing 20 mM acetate pH 6.0, 150 mM NaCl and 0.04% digitonin was incubated with 0.5 mM ML-SA1 (Tocris Bioscience, dissolved in DMSO as a 20 mM stock) on ice for 1 h before grid preparation and freezing. The grids were imaged with a 300 keV Titan Krios electron microscope (FEI) with a Gatan K2 Summit direct electron detector (Gatan). Data were collected at 1 Å per pixel with a dose rate of 8 electrons per physical pixel per second. Images were recorded for 10 s exposures in 50 subframes to give a total dose of 80 electrons per Å2.
Ml sa1
Manufactured by Bio-Techne
Sourced in United States
The ML-SA1 is a laboratory instrument designed for the analysis of small molecules. It features high sensitivity and specificity for the detection and quantification of various analytes. The core function of the ML-SA1 is to provide accurate and reliable analytical data for researchers and scientists working in fields such as chemistry, biology, and pharmaceuticals.
Automatically generated - may contain errors
Lab products found in correlation
Paxilline, by Cayman Chemical (2 mentions)
Ml si1, by Enzo Life Sciences (2 mentions)
Ns1619, by Merck Group (2 mentions)
Bapta am, by Bio-Techne (2 mentions)
K2 summit direct electron detector, by Ametek (2 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (2 mentions)
R1.2 1 3 400 mesh au holey carbon grids, by Quantifoil (2 mentions)
Ionomycin, by Cayman Chemical (2 mentions)
Alexa 488 goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Egta am, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse m csf carrier free, by BioLegend (1 mentions)
4 methylumbelliferyl n acetyl β d glucosaminide, by Merck Group (1 mentions)
Nelfinavir, by Merck Group (1 mentions)
Abacavir, by Merck Group (1 mentions)
Lamivudine, by Merck Group (1 mentions)
Nevirapine, by Merck Group (1 mentions)
Ritonavir, by Merck Group (1 mentions)
Darunavir, by MedChemExpress (1 mentions)
Dolutegravir, by MedChemExpress (1 mentions)
Elvitegravir, by MedChemExpress (1 mentions)
12 protocols using ml sa1
1
Cryo-EM of ML-SA1-Bound Protein
2
Activation of TRPML1 with ML-SA1
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3
Cryo-EM Imaging of Lipid-Bound Proteins
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A protein sample was added to Quantifoil R1.2/1.3 400 mesh Au holey carbon grids (Quantifoil), blotted with Vitrobot Mark IV (FEI), and frozen in liquid ethane. For PtdIns(3,5)P2 or PtdIns(4,5)P2 bound protein, the protein in a buffer containing 20 mM sodium acetate, pH 5.0, 150 mM NaCl and 0.06% digitonin was incubated with 0.2 mM PtdIns(3,5)P2 or PtdIns(4,5)P2 (Echelon, dissolved in H2O as a 10 mM stock) on ice for 1 h before grid preparation and freezing. For PtdIns(3,5)P2/ML-SA1 bound protein, the protein in a buffer containing 20 mM sodium acetate, pH 5.0, 150 mM NaCl and 0.06% digitonin was incubated with 0.2 mM PtdIns(3,5)P2 and 0.3 mM ML-SA1 (Tocris Bioscience, dissolved in DMSO as a 20 mM stock) on ice for 1 h before grid preparation and freezing. The grids were imaged with a 300 keV Titan Krios (FEI) with a Gatan K2 Summit direct electron detector (Gatan). The data were collected at 1.07 Å per pixel with a dose rate of eight electrons per physical pixel per second. Images were recorded for 12-s exposure in 30 subframes to give a total dose of 84 electrons per Å2.
4
Immunofluorescence Staining of Slo1 and Lamp1
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The following primary antibodies were used for immunofluorescence staining: anti-Slo1 (UC Davis/NIH NeuroMab Facility, Davis, CA or Abcam, Cambridge, UK), anti-Lamp1 (H4A3, 1D4B, Developmental Studies Hybridoma Bank, USA), Alexa 488 goat anti-rat antibody, Alexa 488 goat anti-rabbit antibody, Alexa 546 goat anti-mouse antibody and Texas Red goat anti-mouse antibodies were purchased from Invitrogen (Invitrogen, USA). The following chemicals were also used: Paxilline (Cayman Chemical Company, USA); TEA (Sigma, USA); ML-SA1 (Tocris Bioscience, UK); ML-SI1 (Enzo Life Sciences Inc, USA); NS1619 (Sigma, USA); BAPTA-AM (Tocris Bioscience, UK); EGTA-AM (Invitrogen, USA); Recombinant Mouse M-CSF (carrier-free) (BioLegend, USA); 4-Methylumbelliferyl N-acetyl-β-d-glucosaminide (Sigma, USA).
5
Evaluating Anti-HIV and Anti-Alzheimer's Compounds
6
Modulating Cell Contractility and Ion Channels
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Blebbistatin: cells were incubated with 70 µm para-nitro blebbistatin (Optopharma) for 40 minutes at 37 °C in RPMI media before the experiments, unless otherwise stated. MLSA1: cells were incubated with 10 µm mucolipin synthetic agonist 1 (MLSA1, TOCRIS) for 30 minutes at 37 °C in RPMI media before the experiments. Dimethyl sulfoxide (DMSO) was used as a control.
7
Pharmacological Modulators of Cellular Processes
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BafA1, PIK-III, and AZD8055 were purchased from Selleckchem. ML-SA1 and MK6-83 were purchased from Tocris. Monensin, nigericin, salinomycin, valinomycin, and LLoMe were purchased from Sigma-Aldrich. C8 is available for purchase through ChemShuttle (catalog no. 187417).
8
Ion Channel Activation and Modulation
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MLSA1 (4746; Tocris): In all cases, the agonist was added immediately before registration at a final concentration of 40 µM.
Chloroquine (C6628; Sigma): In all cases, culture medium supplemented with 25 µM Chloroquine was used during hemocyte attachment and was kept during recording. Thus, recording began 1 h after drug exposure.
Thapsigargin (T9033; Sigma): The initial culture medium supplemented with 25 µM Chloroquine during attachment was changed to 1 µM Thapsigargin-containing medium immediately before recording.
Chloroquine (C6628; Sigma): In all cases, culture medium supplemented with 25 µM Chloroquine was used during hemocyte attachment and was kept during recording. Thus, recording began 1 h after drug exposure.
Thapsigargin (T9033; Sigma): The initial culture medium supplemented with 25 µM Chloroquine during attachment was changed to 1 µM Thapsigargin-containing medium immediately before recording.
9
Dextran and ion channel modulation
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10
Induction of Lysosomal Damage
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For induction of lysosomal damage, all cells were incubated in complete DMEM or MEF medium with 10% FBS, supplemented with DMSO (vehicle, 0.33%v/v, Millipore Sigma) or L-leucyl-L-leucine dimethyl ester (LLOMe, Sigma) at 1mM (unless otherwise noted) for times indicated. DMSO and LLOMe were equilibrated in fresh, pre-warmed (37°C) complete DMEM with 10% FBS prior to addition to cells.
All other treatments were carried out in complete DMEM or MEF medium with 10% FBS. Bafilomycin-A1 (Sigma) was dissolved in DMSO used at a final concentration of 200nM for 2 hours. Ammonium chloride (Fisher) was dissolved in sterile water and used at a final concentration of 20mM for 1 hour. ML-SA1 (Tocris) was dissolved in DMSO and used at a final concentration of 20μM for 5-30 minutes. For calcium chelation experiments, cells were pre-treated with 10μM BAPTA-AM (Tocris) for 30 minutes. Then, cells were treated with fresh medium containing either DMSO or LLOMe in the presence of an additional 10μM BAPTA-AM for indicated times.
All other treatments were carried out in complete DMEM or MEF medium with 10% FBS. Bafilomycin-A1 (Sigma) was dissolved in DMSO used at a final concentration of 200nM for 2 hours. Ammonium chloride (Fisher) was dissolved in sterile water and used at a final concentration of 20mM for 1 hour. ML-SA1 (Tocris) was dissolved in DMSO and used at a final concentration of 20μM for 5-30 minutes. For calcium chelation experiments, cells were pre-treated with 10μM BAPTA-AM (Tocris) for 30 minutes. Then, cells were treated with fresh medium containing either DMSO or LLOMe in the presence of an additional 10μM BAPTA-AM for indicated times.
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