Peracetylated 2-Acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose (4F-GlcNAc) and 2-Acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-galactopyranose (4F-GalNAc) were from previous studies 20 (link), 27 (link). Peracetylated 1-(Naphthalen-2-ylmethanol) 2-Acetamido-3,4,6-tri-O-Acetyl-2-Deoxy-1-Thio-β-D-Glucopyranoside, GlcNAc-β-S-NAP (abbreviated SNAP), and corresponding peracetylated O-glycoside GlcNAc-β-O-NAP (abbreviated ONAP) were recently described 21 (link). Azido-derivatized carbohydrates ManNAz (N-azido acetyl-mannosamine tetraacylated), GalNAz (N-azido acetyl-galactosamine tetraacylated) and GlcNAz (N-azido acetyl-glucosamine tetraacylated) were purchased from ThermoFisher. Nucleotide-sugars and nucleotides were obtained from either Carbosynth (Compton, UK) or Sigma-Aldrich (St. Louis, MO). All other chemicals were from either ThermoFisher or Sigma unless otherwise mentioned.
Galnaz
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
GalNAz is a chemical probe used in the investigation of cellular glycosylation processes. It functions by being metabolically incorporated into glycoproteins, allowing for the visualization and analysis of glycan structures and their dynamics within living cells.
Automatically generated - may contain errors
Lab products found in correlation
Glcnaz, by Thermo Fisher Scientific (2 mentions)
Mannaz, by Thermo Fisher Scientific (1 mentions)
Tamra glycoprotein detection kit, by Thermo Fisher Scientific (1 mentions)
Eclipse 90i fluorescence microscope, by Nikon (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Operetta high content imaging system, by PerkinElmer (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Complete mini protease inhibitor tablet, by Roche (1 mentions)
Streptavidin agarose, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
5 protocols using galnaz
1
Synthetic Carbohydrate Reagents for Biochemical Studies
2
Visualizing Mucin Glycosylation in Mice
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A total volume of 0.5 mL per mouse of the Click-iT reagent N-azidoacetylgalactosamine (GalNAz) (C33365, Thermo Fischer Scientific) was prepared by dissolving 2.6 mg of GalNAz in 100 µL Dimethyl sulfoxide (A3672, AppliChem GmbH) and diluted in 1 mL in PBS (0.15 mol/L NaCl, 5 mmol/L sodium phosphate buffer, pH 7.4). The injections were given intraperitoneally. Mice were sacrificed 3 h after the GalNAz injection. Paraffin-embedded samples were dewaxed, hydrated, and washed in PBS. Membrane sections were incubated with 20 µL of the reaction mix from the tetramethylrhodamine (TAMRA) glycoprotein detection kit (C10410, Thermo Fischer Scientific) and incubated at room temperature for 2 h, followed by one wash in PBS. The sections were mounted with DAPI containing Prolong Gold anti-fade reagent (P36935, Thermo Fisher Scientific) to visualize DNA (nuclei). The intensity and localization of freshly produced mucins were evaluated in a blinded fashion at 40x magnification on an Eclipse 90i fluorescence microscope (Nikon). Due to that co-stain with antibody against Muc2 affected the TAMRA intensity; goblet cells were identified based on cellular morphology.
3
Metabolic Labeling and Imaging of Cells
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Sugar ManNAz, GalNAz, GlcNAz and Alexa Fluor 488 DIBO Alkyne (DIBO488 ) were purchased from ThermoFisher. Live cells were treated with 50 μM sugars at either 2, 6, 24 and 48 hours. Afterwards, cells were washed and were labelled with 15 μM DIBO488 and 5 μM COA-1 for 1 h at 37 °C, followed by counterstaining with nuclear tracker, DRAQ5 . Cells were imaged using the Operetta High-Content Imaging System (PerkinElmer) with a 40× objective lens.
4
Labeling Anaerobic Bacterium with AF647-DIBO
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ZY-312 was cultured for 65 h at 37 °C under anaerobic conditions (80% N2, 10% H2, 10% CO2) in an anaerobic glove box in basal peptone-yeast broth (OXOID, United Kingdom) with GalNAz (Thermofisher, United States) at a final concentration of 100 μM. Bacterium were spun down and washed three times in 1 × phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA) when the OD600 value ranged from 0.7 to 1.4. The bacteria pellet was resuspended in 10 ml PBS supplemented with 1% BSA (Sigma, Germany) and 20 mM AF647-DIBO (100 μl per vial) and each vial of the bacterium suspension was kept separately. To determine the optimal incubation time, bacteria suspensions were incubated for 3 h, 5 h, 7 h, and overnight in dark on a shake flask. After incubation, bacteria were pelleted and washed five times with PBS supplemented with 3% BSA. Finally, the bacteria were resuspended either in PBS for in vivo tracing or in 90% glycerol for fluorescent microscope observation.
5
Biotinylation and Immunoprecipitation of CSF3R Variants
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