The membrane fraction was harvested according to the standard procedure53 (link) with minor modification. In brief, CCA cells were homogenized in 20 mM HEPES-KOH, pH 7.4 containing 0.25 M sucrose and 1:100 protease inhibitor, using probe sonication (25 Amplitude, pulsed on 5 sec and off 10 sec for 5 times). Cell nuclei were fractionated by centrifugation at 2,000 xg, for 10 min. Then, the membrane fraction was separated by 3 times of ultra-centrifugation at 200,000 xg, 45 min. The final pellet was collected as the membrane fraction and total N-glycans were released using 1,000 U of PNGase-F at 37°C in a microwave reactor (CEM Corporation, Matthews, NC) at 20 watts for 10 min. The released N-glycans were enriched and purified by porous graphitized carbon solid-phase extraction as described previously.48 (link) The unbound fraction was first eluted with 9 ml of pure water and the bound N-glycan fraction was eluted with 4 ml of 40% ACN and 0.1% TFA in pure water.
Pngase f
Manufactured by CEM Corporation
PNGase-F is an enzyme that catalyzes the removal of asparagine-linked glycans from glycoproteins. It is a commonly used tool in the analysis and study of glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pngase f
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N-Glycan Profiling from Membrane Fractions
2
N-Glycan Extraction from Membrane Fraction
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The membrane fraction was harvested according to the standard procedure [53 (link)] with minor modification. In brief, CCA cells were homogenized in 20 mM HEPES-KOH, pH 7.4 containing 0.25 M sucrose and 1:100 protease inhibitor, using probe sonication (25 Amplitude, pulsed on 5 s and off 10 s for 5 times). Cell nuclei were fractionated by centrifugation at 2000 × g, for 10 min. Then, the membrane fraction was separated by 3 times of ultra-centrifugation at 200,000 × g, 45 min. The final pellet was collected as the membrane fraction and total N-glycans were released using 1000 U of PNGase-F at 37 °C in a microwave reactor (CEM Corporation, Matthews, NC) at 20 watts for 10 min. The released N-glycans were enriched and purified by porous graphitized carbon solid-phase extraction as described previously [48 (link)]. The unbound fraction was first eluted with 9 ml of pure water and the bound N-glycan fraction was eluted with 4 ml of 40% ACN and 0.1% TFA in pure water.
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