PNGase F N-glycan Releasing Kit

PNGase F is an amidase that cleaves the amide bond between the GlcNAc1 residue of anN-glycan and the underlying asparagine residue. Only the pentasaccharide core of an N-glycan ispictured here for clarity. The pentasaccharide can be extended at R1, R2 and R3 positions. R1 can bean alpha -6 fucose but not an alpha -3 fucose. R2 and R3 can be any of common monosaccharides oroligosaccharides. Fluorescent tags can be introduced to R1, R2, and R3 with no obvious change onthe sensitivity to PNGase F digestion.

Deglycosylation of Rb (A) and Cy5-Rb (B) with PNGase F N-glycan Releasing Kit(R&D Systems®, Catalog # EA006) using both the native and denaturing assay protocols. >95%deglycosylation was achieved with 1000 ng and 1.6 ng of PNGase F under native and denaturingconditions, respectively. Digested samples were separated on 17% SDS gel. A. Rb (1 μg perdigestion) was digested with indicated amounts of PNGase F and imaged with silver staining.B. Cy5-Rb (0.2 μg for digestion) was digested with indicated amounts of PNGase F and imaged byFluorchem R/M. Labeling on RNase B was introduced by serial treatment with Recombinant HumanN-Acetylglucosaminyltransferase1/MGAT1, CF (R&D Systems®,Catalog # 8334-GT)/UDP-GlcNAc,Recombinant Human B4GalT1 Protein, CF (Catalog # 3609-GT)/UDP-Gal, and Recombinant HumanST6GAL1 (aa44-406) Protein, CF (Catalog # 7620-GT)/CMP-Cy5-Sialic Acid (Catalog # ES302)(Wu, Z. et al. (2020) Glycobiology 30:970). Denaturing increased the PNGase F sensitivity by 500-fold.

Deglycosylation of Cy5-RBD with PNGase F N-glycan Releasing Kit (R&D Systems®, Catalog # EA006) using both the native and denaturing assay protocols. Cy5-RBD (1 µg per digestion) was treated with indicated amounts of PNGase F in 20 µL under both denaturing and native conditions at 37 °C for 60 minutes then separated on 17% SDS gel and imaged by trichloroethanol (TCE) staining (top panel, only proteins were visible, and the lower portion wascropped out) and fluorescent imaging with Fluorchem R/M (lower panel). >95% deglycosylation was achieved with 200 ng and 1.6 ng of PNGase F under native and denaturing conditions, respectively.

Deglycosylation of Cy5-ACE-2 with PNGase F N-glycan Releasing Kit (R&D Systems®, Catalog # EA006) using both the native and denaturing assay protocols Cy5-ACE-2. (1 µg for each digestion) was treated with indicated amounts of PNGase F in 20 µL under both denaturing and native conditions at 37 °C for 30 minutes then separated on 17% SDS gel and imaged by TCE staining (top panel, only proteins were visible, and the lower portion was cropped out) andfluorescent imaging with Fluorchem R/M (lower panel). Multiple bands are visible for the released glycans suggesting the complicated nature of the glycosylation on ACE-2. >90% deglycosylation was achieved with 2000 ng and 0.2 ng of PNGase F under native and denaturing conditions, respectively

pH profile of PNGase F on Cy5-RBD with PNGase F N-glycan Releasing Kit (R&D Systems®, Catalog # EA006). Denatured Cy5-RBD (1 µg per digestion) was treated with 5 ng of PNGase F at indicated pH in 20 µL at 37 °C for 60 minutes then separated on 17% SDS gel and imaged by TCE staining (top panel, only proteins were visible, and the lower portion was cropped out) and fluorescent imaging with Fluorchem R/M (lower panel). PNGase F showed activity from pH 5.0 to 10 with complete glycan removal between pH 6.0 to pH 8.5.

Mass spectrum of PNGase F treated fetal bovine RNase B. Native RNase B of different glycoforms with high-mannose glycans, Man-5 to Man-9, are indicated as Rb-Man5 to Rb-Man9 respectively.Upon PNGase F treatment under native condition, majority of these glycans were removed and resulted in deglycosylated RNase B (Rb). Mass spectrometry was performed using a Thermo Scientific Q Exactive HF system connected to a Thermo Vanquish LC. Samples were separated on a C4 column then analyzed using full scan at 240,000 resolution in intact mode. Data was analyzed using Thermo BioPharma Finder software.

• Reactivity: All-Multi

PNGase F N-glycan Releasing Kit Summary

• Additional Information: 100 Test per Kit
• Inter-Assay: See PDF Datasheet for details
• Intra-Assay: See PDF Datasheet for details
• Spike Recovery: See PDF Datasheet for details
• Sample Volume: See PDF Datasheet for details

Packaging, Storage & Formulations

• Storage: Store the unopened product at -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for PNGase F N-glycan Releasing Kit

• PNGase F
• PNGF