For experiments reported in SI Appendix, Fig. S1 A–C , the cGAMP degradation activity assay (20 μL) for cells containing 50% cell lysate, cGAMP (1 μM, with trace [32P] cGAMP spiked in), and standard ENPP1 activity buffer (50 mM Tris pH 9, 250 mM NaCl, 0.5 mM CaCl2, 1 μM ZnCl2) took place in room temperature. For experiments reported in SI Appendix, Fig. S6 A and B , the cGAMP degradation activity assay for mouse organs (30 μL) containing 75% organ lysate (100 mg/mL), 5 μM cGAMP, and PBS took place in 37 °C. Last, cGAMP degradation activity assay (20 μL) for mouse serum containing 50% serum, 5 μM cGAMP and physiological ENPP1 activity buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM CaCl2, and 10 μM ZnCl2) took place in 37 °C. At indicated times, 1 μL aliquots of the reaction were quenched by spotting on HP-TLC silica gel plates (Millipore). The TLC plates were run in mobile phase (85% ethanol, 5 mM NH4HCO3) and exposed to a phosphor screen (GE BAS-IP MS). Screens were imaged on a Typhoon 9400 scanner and the 32P signal was quantified using ImageJ. The sample size and statistical tests of computation are indicated in the respective figure legend.
Hptlc silica gel plates
Manufactured by Merck Group
Sourced in Germany
HPTLC silica gel plates are a type of thin-layer chromatography (TLC) substrate used for the separation and analysis of complex mixtures. These plates are coated with a thin layer of silica gel, which acts as a stationary phase. Samples are applied to the plate, and the mobile phase is then allowed to migrate through the silica gel, facilitating the separation of the components in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Bas ip ms, by GE Healthcare (2 mentions)
Tlc plate heater 3, by CAMAG (2 mentions)
Chromatogram immersion device 3, by CAMAG (1 mentions)
Automatic tlc sampler 4, by CAMAG (1 mentions)
Reprostar 3, by CAMAG (1 mentions)
Phospho foxo1, by Cell Signaling Technology (1 mentions)
Antibody against hsp70, by Enzo Life Sciences (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Hplc grade methanol, by Merck Group (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Chenodeoxycholic acid, by Merck Group (1 mentions)
Sulfosalicylic acid, by Merck Group (1 mentions)
Qiashredder kit, by Qiagen (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
6 protocols using hptlc silica gel plates
1
Quantification of cGAMP Degradation
2
cGAMP Degradation Activity Assay
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For experiments reported in Figure S1A –C , cGAMP degradation activity assay (20 μl) for cells containing 50% cell lysate, cGAMP (1 μM, with trace [32P] cGAMP spiked in), and standard ENPP1 activity buffer (50 mM Tris pH 9, 250 mM NaCl, 0.5 mM CaCl2, 1 μM ZnCl2) took place in room temperature. For experiments reported in Figure S6A –B , cGAMP degradation activity assay for moues organs (30 μl) containing 75% organ lysate (100 mg/mL), 5 μM cGAMP, and PBS took place in 37°C. Lastly, cGAMP degradation activity assay (20 μl) for mouse serum containing 50% serum, 5 μM cGAMP and physiological ENPP1 activity buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM CaCl2, 10 μM ZnCl2) took place in 37°C. At indicated times, 1 μl aliquots of the reaction were quenched by spotting on HP-TLC silica gel plates (Millipore). The TLC plates were run in mobile phase (85% ethanol, 5 mM NH4HCO3) and exposed to a phosphor screen (GE BAS-IP MS). Screens were imaged on a Typhoon 9400 scanner and the 32P signal was quantified using ImageJ. The sample size and statistical tests of computation are indicated in respective figure legend.
3
Lipid Profiling by HPTLC Imaging
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Aliquots of 1 µL of each seed lipid extract were applied to the 20 cm×10 cm HPTLC silica gel plates (Merck, Darmstadt, Germany) as 6-mm band by using Automatic TLC sampler 4 (ATS4; CAMAG, Muttenz, Switzerland). Plates were developed in the saturated twin trough chamber up to a migration distance of 90 mm, using four mobile phases: V(chloroform):V(methanol):V(acetic acid)=90:10:1 up to 25 mm, V(n-hexane):V(diethylether):V(acetone)=60:40:5 up to 70 mm, V(n-hexane):V(diethylether)=97:3 up to 85 mm and 100% n-hexane up to 90 mm. The developed plates were dried in a stream of warm air and dipped in post-chromatographic derivatization solution of cerium ammonium molybdate for 1 s with an immersion speed of 3.5 cm/s, using Chromatogram Immersion Device III (CAMAG). Derivatized plates were heated for 5 min at 110 °C on TLC Plate Heater III (CAMAG). Images were captured at 366 nm with DigiStore 2 device image analysing system in conjunction with Reprostar 3 (CAMAG). Four apertures with exposure time of 30 ms and frame of 2 mm were applied. The photos were stored as TIF files for further image processing.
4
Investigating Cellular Stress Responses
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Penicillin-streptomycin was obtained from Lonza (Walkersville, MD, USA). The EZ-Cytox Cell Viability Kit was purchased from Daeil Lab (Seoul, Korea). LA, N-acetyl-l -cysteine (NAC), 2′,7′-dichlorofluorescein diacetate (DCF-DA), ortho-phthaldialdehyde (OPA), chloroform, and trypsin-EDTA solutions were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from Gibco Life Technologies Inc. (Rockville, MD, USA). Antibody against HSP70 was purchased from Enzo Life Sciences (Aargau, Switzerland). Antibodies against Bcl-2, Bax, phospho-FoxO1, β-actin, anti-rabbit IgG, and anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SOD1 and MuRF1 were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Vectashield mounting medium containing DAPI was purchased from Vector Laboratories (Burlingame, CA, USA). HPLC-grade methanol and HPTLC silica gel plates were purchased from Merck (Darmstadt, Germany). Ceramide, sphingomyelin, dihydrosphingomyelin, and sphingolipid ceramide N-deacylase (SCDase) were purchased from Avanti Polar Lipid Inc. (Alabaster, AL, USA). All other chemicals were commercially available.
5
Techniques for Protein Kinase C Activation and Inhibition
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Paraformaldehyde, biotin, bovine serum albumin (BSA), phorbol 12-myristate 13-acetate (protein kinase C (PKC) activator), Ro 31-0432 (PKC inhibitor), chenodeoxycholic acid (CDCA), diaminobenzidine tetrahydrochloride tablets, NADPH, and sulfosalicylic acid were supplied by Sigma-Aldrich (St. Louis, MO, USA); avidin was obtained from Fluka (Buchs, Switzerland), the cholera toxin B subunit (CTB) peroxidase conjugated came from List Biological Laboratories (CA, USA), and the HPTLC silica-gel plates came from Merck (Darmstadt, Germany). Cell plates were supplied by Corning (NY, USA). The TaqMan® Gene Expression Master Mix, High-Capacity RNA-to-cDNA Kit, and the TaqMan Gene Expression Assay kit for mouse and human genes were obtained from Life Technologies (Carlsbad, CA, USA). The QIAshredder kit and RNeasy Plus Mini Kit were supplied by Qiagen (USA). All other chemicals were purchased locally from Penta (Prague, Czech Republic).
6
Quantitative TGA Analysis of Lipids
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The TGAs analysis was performed on Camag TLC Scanner 3. The 2 µL of lipid extract was applied to 20 × 20 cm HPTLC silica gel plates (Art. 105641, Merck) as 6-mm band by using an Automatic TLC sampler 4 (ATS4, CAMAG, Muttenz, Switzerland). The ascending chromatography was performed in the CAMAG twin-trough chamber using four mobile phases: chloroform:methanol:acetic acid (90:10:1, v/v/v) up to 25mm, n-hexane:diethyl-ether:acetone (60:40:5, v/v/v) up to 70 mm, n-hexane:diethyl-ether up to 85 mm, and 100% n-hexane up to 90mm. Before the analysis, the chambers were saturated for 30 min. After the last mobile phase development, the plates were dried in the dark. Derivatization was performed by spraying with a mixture of methanol and concentrated sulphuric acid (9/1, v/v). Derivatized plates were heated at 80 °C on TLC Plate Heater III (CAMAG) until chromatographic zones become visible, followed by scanning in CAMAG TLC Scanner 3. TGAs concentration was determined based on the intensity of the standards and samples.
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