Auxin

Manufactured by Thermo Fisher Scientific

Sourced in United States

Auxin is a laboratory equipment used for the measurement and analysis of various biological and chemical samples. It utilizes advanced technology to provide accurate and reliable data. The core function of Auxin is to facilitate precise quantification and characterization of the samples under investigation.

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Lab products found in correlation

Dynabeads protein g beads, by Thermo Fisher Scientific (1 mentions) Auxin, by Merck Group (1 mentions) Rabbit anti h3, by Abcam (1 mentions) Rabbit anti h3k14ac, by Merck Group (1 mentions) Cell culture flasks, by Sarstedt (1 mentions) β estradiol, by Merck Group (1 mentions)

12 protocols using auxin

Auxin-mediated priming of N. parisii immunity

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auxin plates were prepared by adding auxin stock solution [400 mM auxin (Alfa Aesar) in ethanol] to NGM, for a final concentration of 200 μM auxin, immediately before pouring plates. Control plates were prepared by adding ethanol to NGM, for a final concentration of 0.15%. auxin plates were stored in the dark at 4°C and used within 1 month.
Embryos obtained from bleaching gravid adults were plated on auxin or ethanol control plates following M9 washes. 10× OP50-1 was added to plates 18 to 24 hours after plating to allow embryos to hatch and synchronize. Worms were bleached 72 hours after L1 arrest and F1 immunity to N. parisii tested as described in the “Basic infection and priming assays” section.

Willis A.R., Zhao W., Sukhdeo R., Wadi L., El Jarkass H.T., Claycomb J.M, & Reinke A.W. (2021). A parental transcriptional response to microsporidia infection induces inherited immunity in offspring. Science Advances, 7(19), eabf3114.

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Rapid Depletion of HAT Activity

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All 12 HAT-AID fusion strains (Supplementary Table 1) were generated to rapidly deplete specific HAT activity and the wild type cells were cultured in SDC medium without inositol until OD₆₀₀ 0.3. At this point, cultures were split in two and auxin (I2886, Sigma-Aldrich) was added to one of the tubes for each sample to a final concentration of 500 μM. Cells were incubated with auxin for 1 h 30 min followed by immobilization on ConA-coated Nunc 96 well Optical-Bottom plates (164588, ThermoFisher). Imaging and nucloc analysis were performed as previously described^{12 (link)} for the Statistical Mapping of the INO1 locus wild type and the GRS mutants. Western Blots were performed on HAT-AID fusion strains to verify the degradation of the corresponding HAT once the auxin was added to growth medium.
ChIP-qPCR experiments for H3K14ac and H3 in the AID strains were performed in three biological replicates similarly as described before for the H3-FLAG with modifications for the sample preparation and the IP procedures. In this case, cells were grown under the same conditions as described for the AID experiments before crosslinking. For the IP protocols, 50 μL of Dynabeads™ Protein G beads (ThermoFisher Scientific) pre-coupled to 2 µL of rabbit anti-H3K14ac (Millipore) or 2 µg of rabbit anti-H3 (Abcam) were used per IP sample.

González L., Kolbin D., Trahan C., Jeronimo C., Robert F., Oeffinger M., Bloom K, & Michnick S.W. (2023). Adaptive partitioning of a gene locus to the nuclear envelope in Saccharomyces cerevisiae is driven by polymer-polymer phase separation. Nature Communications, 14, 1135.

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Acute Knockdown of Kinesin-1 in DA9

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To acutely knock down kinesin-1/unc-116 in DA9, young L4 animals were cultured on NGM plates containing 1 mM Auxin (Thermo-scientific, #A10556.14) for 5 hours before imaging. Animals of the same stage cultured on NGM plates lacking Auxin were served as the controls.

Wu Y., Ding C., Weinreb A., Manning L., Swaim G., Yogev S., Colón-Ramos D.A, & Hammarlund M. (2023). Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport. bioRxiv.

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UV Crosslinking and Auxin Depletion Protocols

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For UV crosslinking, strains were grown overnight in –TRP synthetic dropout medium with 2% glucose, diluted to OD₆₀₀ 0.05 in 3L media and grown to OD₆₀₀ 0.5 at 30°C. Cells were collected by filtration, washed with –TRP medium containing 2% glycerol and 2% ethanol (GE), and transferred to fresh –TRP medium containing GE. Cells were then grown for an additional 4 or 8 min before UV crosslinking. As a control, a second culture was grown in parallel and crosslinked while still in glucose containing medium (‘0’ time point), or filtered and transferred to fresh glucose-containing medium (‘mock’).
Addition of auxin (Indole-3-acetic acid from Alfa Aesar) impaired cell growth in standard SD media but not in Kaiser SD, which was therefore used for Nrd1 depletion experiments. Cells were grown overnight in 2% glucose medium lacking methionine, diluted to OD₆₀₀ 0.05 in 1 L culture and grown to OD₆₀₀ 0.3 at 30°C. auxin and methionine were added to final concentrations of 1 mM and 670 μM, respectively, and cells were cultured for an additional 90 min (OD₆₀₀ ∼0.5) prior to harvesting.

Bresson S., Tuck A., Staneva D, & Tollervey D. (2017). Nuclear RNA Decay Pathways Aid Rapid Remodeling of Gene Expression in Yeast. Molecular Cell, 65(5), 787-800.e5.

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Auxin Application to Adult Worms

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Auxin (indole-3-acetic acid) was purchased from Alfa Aesar. A 100× stock Auxin solution (0.4 M) was made by dissolving 0.7 g of Auxin in 10 ml of 100% ethanol. A mixture of 25 μl of stock Auxin solution and 225 μl of distilled water was added to plates containing 1-day-old adult worms.

Joseph B.B., Edeen P.T., Meadows S., Binti S, & Fay D.S. (2022). An unexpected role for the conserved ADAM-family metalloprotease ADM-2 in Caenorhabditis elegans molting. PLoS Genetics, 18(5), e1010249.

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Inducing Infertility in C. elegans

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Synchronous larvae were washed twice in M9 buffer, counted and adjusted to 10 larvae per 10 μl. Nematodes were raised in cell culture flasks (Sarstedt, Nürmbrecht, Germany) or OP50 spread NGM plates. OP50-NGM was given as a standardized food source with a volume 4.4-fold of the larvae containing M9 solution used. L1 larvae were maintained under continuous shaking at 20°C, reaching adulthood within 3 days.
PX627 were treated with 1 mM indole-3-acetic acid (auxin, Alfa Aesar, Haverhill, MA, USA) to induce infertility during the L3 stage [12 (link), 13 (link)]. Wild-type N2 nematodes were treated with 100 μM 5-fluoro-2´-deoxyuridine (FUdR) or M9 control after reaching young adulthood. PX627 were also treated with M9 control 48 h prior to the assessment on day-2. The day upon which the nematodes reached young adulthood was defined as day 1. To ensure ad libitum food supply until day 10, at day-2 and day-6, old OP50-NGM was discarded after sedimentation of gravid nematodes and fresh OP50-NGM, with effectors incorporated, was given. For the measurements on day-2, FUdR- and auxin-treated young controls and untreated nematodes were co-assessed, whereas at day-10, only sterilized and unfertilized animals could be assessed.

Dilberger B., Baumanns S., Spieth S.T., Wenzel U, & Eckert G.P. (2020). Infertility induced by auxin in PX627 Caenorhabditis elegans does not affect mitochondrial functions and aging parameters. Aging (Albany NY), 12(12), 12268-12284.

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Auxin Preparation for Seeded Plates

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400 mM auxin in EtOH (indole-3-acetic acid, Alfa Aesar) was used to make 4 mM auxin-containing seeded, unseeded or assay plates. Plates were kept wrapped in aluminum foil to prevent light exposure.

Takeishi A., Yeon J., Harris N., Yang W, & Sengupta P. (2020). Feeding state functionally reconfigures a sensory circuit to drive thermosensory behavioral plasticity. eLife, 9, e61167.

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Auxin-induced Worm Development

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Worms of the indicated genotype were picked as L4s on OP50-seeded NGM plates containing 1 mM 3-Indoleacetic acid (i.e. auxin, Alfa Aesar A10556-22) dissolved in absolute ethanol and maintained at 20°C for the specified times.

Hicks T., Koury E., McCabe C., Williams C., Crahan C, & Smolikove S. (2022). R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline. Nucleic Acids Research, 50(14), 8041-8059.

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Auxin Exposure in Adult Animals

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1-day-old adult animals were transferred to NGM plates containing 1 mM auxin (indole-3-acetic acid) (#A10556.06, Alfa Aesar) for indicated times. The 1 mM auxin-containing agar plates were prepared 24 h before use from a freshly made 800 mM stock solution in 99.5% ethanol and stored in the dark. An equivalent amount of ethanol was added to the control plates.

Raj D., Kraish B., Martikainen J., Podraza-Farhanieh A., Kao G, & Naredi P. (2023). Cisplatin toxicity is counteracted by the activation of the p38/ATF-7 signaling pathway in post-mitotic C. elegans. Nature Communications, 14, 2886.

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Auxin-Mediated Worm Development

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Auxin (indole-3-acetic acid) was purchased from Alfa Aesar. A 100× stock Auxin solution (0.4 M) was made by dissolving 0.7 g of Auxin in 10 ml of 100% ethanol. A mixture of 25 μl of stock Auxin solution and 225 μl of autoclaved deionized water was added to each plate containing day-1 adult worms.

Joseph B.B., Naslavsky N., Binti S., Conquest S., Robison L., Bai G., Homer R.O., Grant B.D., Caplan S, & Fay D.S. (2023). Conserved NIMA kinases regulate multiple steps of endocytic trafficking. PLOS Genetics, 19(4), e1010741.

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