New England Biolabs, P0706S, Remove-iT® PNGase F

Remove-iT Related Categories Endoglycosidases,, Proteome Analysis Applications Expression Systems,, Glycan Sequencing,, Proteomics, Specification Unit Definition One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 5 μg of DTT denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 2 Incubate at 37°C 1X GlycoBuffer 2 50 mM Sodium Phosphate (pH 7.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 5 mM EDTA pH 7.5 @ 25°C Heat Inactivation 75°C for 10 minutes Molecular Weight Apparent: 41 kDa Unit Assay Conditions 5 µg of RNase B are denatured with 1X DTT at 55°C for 10 minutes. After the addition of 1X GlycoBuffer 2, two-fold dilutions of Remove-iT PNGase F are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE. FAQ Q: What is the tag on PNGase F? A: PNGase F is tagged with a chitin binding domain (CBD). The CBD tag allows for removal from a reaction using chitin beads (NEB #S6651) or chitin magnetic beads (NEB #E8036). Note: there is no cleavage site between the PNGase F and the CBD tag to enable removal of this tag from the PNGase F. Q: I tried deglycosylating my glycoprotein with Remove-iT® PNGase F but did not see removal of the carbohydrate. What could be the problem? A: Do you know how the carbohydrate is attached to the protein? Is it N-, or O-linked (linked to an Asn, or a Ser/Thr)? Remove-iT® PNGase F will only remove carbohydrates attached to an Asn. NEB’s O-Glycosidase (NEB# P0733) will remove core 1 and core 3 O-linked carbohydrates attached to Ser/Thr. If you are not seeing deglycosylation and you know your protein has N-glycans consider adding more enzyme and using longer incubation times. If you have chosen to denature the glycoprotein, be certain to add NP-40 (to protect PNGase F, Recombinant from the SDS in the denaturation step). Outside these reaction conditions there is the possibility that the carbohydrate may be resistant to PNGase F, Recombinant (a rare occurrence). This happens when the core N-acetylglucosamine is modified by an α1-3Fucose (often found in plant and insect proteins). Q: What is the difference between PNGase F and Remove-iT® PNGase F? A: PNGase F and Remove-iT® PNGase F are purified from the same source with identical specificity. Remove-iT PNGase F has been expressed with a chitin binding domain (CBD) tag to allow for easy removal of the enzyme from a reaction. The tag does not change or alter the specificity or activity of the enzyme. If used under denaturing conditions, PNGase F and Remove-iT® PNGase F have the same unit concentration. However, Remove-iT® PNGase F is sold with a modified denaturing unit (DTT only, no SDS) to make it mass spec friendly. Q: What is the difference between Remove-iT® PNGase F and Endo H? A: Remove-iT® PNGase F removes all types of N-linked (Asn linked) glycosylation; high mannose, hybrid, bi, tri, and tetra antennary. You will choose this enzyme if your goal is to remove all N- linked carbohydrates without regard to type. Endo H removes only high mannose and some hybrid types of N-linked carbohydrates. You would choose this enzyme to more closely determine the type of N-linked glycosylation, or if you know that the protein has a carbohydrate sensitive to Endo H. Q: How much Remove-iT® PNGase F should I use to remove my carbohydrate under native or DTT denaturing conditions? A: When the protein is not denatured with SDS, Remove-iT® PNGase F has to work harder to reach the cleavage site of the carbohydrate (because of the secondary and tertiary protein structure). Sometimes extra enzyme and extended incubation times can help but these values are specific to each protein and must be determined empirically. Q: Does Remove-iT® PNGase F work in Urea? A: PNGase F is stable in 2.5M urea at 37C for 24 h and still possesses 40% of its activity in 5 M urea1. 1. Maley, F., et al, Anal Biochem, 180, 195-204 (1989) Q: What are the typical reaction conditions for Remove-iT® PNGase F? A: Typical reaction conditions are as follows: Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Combine 10-20 µg of glycoprotein, 1 µL of 40mM DTT and H20 (if necessary to make a 10 µL total reaction volume. Denature glycoprotein by heating reaction at 55°C for 10 minutes. Make a total reaction volume of 20 µL by adding 2 µL 10X GlycoBuffer 2, H20 and 1-5 µL Remove-iT® PNGase F. Incubate reaction at 37°C for 1 hour. Note: Reactions may be scaled-up linearly to accommodate larger reaction volumes. Q: How do I eliminate Remove-iT® PNGase F from a reaction? A: Remove-iT® PNGase F can be removed from a deglycosylation reaction using NEB’s magnetic chitin beads. Typical reaction conditions for removing 1-5 µL of Remove-iT® PNGase F using magnetic chitin beads are as follows: Materials: Remove-iT® PNGase F (NEB #P0706) Chitin Magnetic Beads (NEB #E8036) Magnetic Separation Rack (NEB #S1506 or NEB #S1509) Pipette 50 µl Chitin Magnetic Beads into an eppendorf tube and place the eppendorf in a magnetic separation rack. Let the magnet attract the chitin beads, then pipette off the liquid supernatant and discard. With the eppendorf on the magnetic separation rack, wash the magnetic chitin beads 2 x 500 µl with 50 mM NH4 Formate pH 4.4 (or buffer of choice). Pipette of the supernatant and discard. Add the deglycosylated glycoprotein sample into the eppendorf with magnetic chitin beads. Rock the deglycosylated glycoprotein sample with the magnetic chitin beads for 10 minutes at 4°C. Place the eppendorf back on the magnetic separation rack, and allow the magnet to attract the chitin beads. Pipette off the supernatant and keep. Wash the magnetic chitin beads 3 x 100 µl with 50 mM NH4 Formate pH 4.4 (or buffer of choice). Pipette of the supernatant from each wash and keep. Combine all supernatants from steps 5 & 6, as these are the deglycosylated glycoprotein. Analyze sample by method of choice. Note - Removal of Remove-iT® PNGase F from the deglycosylation reaction can be scaled up linearly with larger magnetic chitin bead volumes. Q: What is the binding capacity of the Magnetic Chitin Beads used to eliminate Remove-iT® PNGase F? A: The Magnetic Chitin Beads (NEB# E8036) binding capacity is approximately 0.4mg/ml of CBD-tagged protein. This binding capacity is calculated in mg of protein per bed volume of resin. The chitin magnetic beads are a 50:50 slurry. Therefore, 50 μl of slurry will yield 25 μl bed volume of resin, which is enough to remove 1-5 µL of Remove-iT® PNGase F. Q: Is Remove-iT® PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry? A: Remove-iT® PNGase F is supplied in a mass spec friendly, glycerol free storage buffer. Glycoprotein Denaturing Buffer (containing SDS and DTT) is not compatible with Mass Spectrometry applications, and often times hard to remove from the reaction. Therefore, the Remove-iT® PNGase F unit definition is based on a partial denaturation protocol using only DTT, with no SDS making the reaction compatible with downstream HPLC and MS analysis. Q: Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers? A: To simplify workflows and digestions with two or more exoglycosidases, most enzymes are now provided with 10X GlycoBuffer 1. Two exceptions are provided with GlycoBuffer 4. Also, the buffer panel for endoglycosidases has been simplified. The activity of every endo- and exoglycosidase enzyme was evaluated in both its old and new buffer system. All enzymes retain either the same activity, or were found to have greater activity in the new buffer. Some exoglycosidases are still provided with an additional vial of rHSA (rHSA enhances the activity of certain enzymes). As before, some exoglycosidases are still provided with an additional vial of BSA (BSA enhances the activity of certain enzymes). Other components (i.e. Glycoprotein Denaturing Buffer, NP-40, etc.) are still provided when required. Enzyme Product # Old buffer Current buffer Changed? α-N-Acetylgalactosaminidase P0734 G7 GlycoBuffer 1 YES α1-2 Fucosidase P0724 G4 GlycoBuffer 1 YES α1-2,3,4,6 Fucosidase P0748 --- GlycoBuffer 1 --- α1-2,4,6 Fucosidase O p0749 --- GlycoBuffer 1 --- α1-3,4 Fucosidase p0769 --- GlycoBuffer 1 --- α1-2,3 Mannosidase P0729 G6 GlycoBuffer 1 no α1-6 Mannosidase P0727 G2 GlycoBuffer 1 YES α1-2,3,6 Mannosidase p0768 --- GlycoBuffer 4 --- α1-3,4,6 Galactosidase P0747 --- GlycoBuffer 1 ---- α1-3,6 Galactosidase P0731 G6 GlycoBuffer 1 no α2-3 Neuraminidase S P0743 --- GlycoBuffer 1 --- α2-3,6,8 Neuraminidase P0720 G1 GlycoBuffer 1 YES α2-3,6,8,9 Neuraminidase A P0722 --- GlycoBuffer 1 --- β-N-Acetylhexosaminidasef P0721 G2 GlycoBuffer 1 YES β-N-Acetylglucosaminidase P0732 G1 GlycoBuffer 1 YES β-N-Acetylglucosaminidase S P0744 --- GlycoBuffer 1 --- β1-3 Galactosidase P0726 G6 GlycoBuffer 1 no β1-4 Galactosidase S P0745 --- GlycoBuffer 1 --- β1-3,4 Galactosidase p0746 --- GlycoBuffer 4 --- N-Glycan Sequencing Kit E0577 --- GlycoBuffer 1 --- Endo S P0741 G6 GlycoBuffer 1 YES Endo H P0702 G5 GlycoBuffer 3 YES Endo Hf P0703 G5 GlycoBuffer 3 YES Endo F2 p0772 --- GlycoBuffer 4 --- Endo F3 p0771 --- GlycoBuffer 4 --- Endo D P0742 G7 GlycoBuffer 2 no O-Glycosidase P0733 G7 GlycoBuffer 2 no O-Glycosidase & Neuraminidase Bundle E0540 G7 GlycoBuffer 2 no Remove-iT® PNGase F P0706 G7 GlycoBuffer 2 no PNGase F P0704 G7 GlycoBuffer 2 no PNGase F (Glycerol-free) P0705 G7 GlycoBuffer 2 no PNGase F, Recombinant P0708 G7 GlycoBuffer 2 no PNGase F (Glycerol-free), Recombinant P0709 G7 GlycoBuffer 2 no PNGase A p0707 --- Glyco Buffer 3 --- Protein Deglycosylation Mix II p6044 --- Deglycosylation Mix Buffer 1 and 2 --- Rapid PNGase F P0710 --- Rapid PNGase F Buffer ---- Rapid™ PNGase F (non-reducing format) p0711 --- Rapid PNGase F (non-reducing format) Buffer Q: What is a good endoglycosidase substrate? A: For PNGaseF and Protein Deglycosylation Mix we suggest Fetuin (NEB #P6042, supplied with the Protein Deglycosylation Mix). For PNGaseF, Endo H, and Endo Hf, we suggest RNase B (NEB #P7817). For Endo D we suggest Phospholipase A from bee venom. For Endo S we suggest mouse monoclonal IgG (NEB #E8032). For O-glycosidase we suggest p-Nitrophenyl galacto-N-bioside (Galβ1-3GalNAcα1pNP, or Core1-pNP). Alternatively, fetuin can be digested with neuraminidase, and neuraminidase plus O-glycosidase. The changes in molecular weight after glycan removal can be observed by SDS-PAGE. Q: Do detergents inhibit exoglycosidases/endoglycosidases? A: At moderate levels (0.5-1.0% ionic and non-ionic detergents) most of the glycosidases show satisfactory activity or are unaffected. Exceptions are: PNGase F (all formulations), O-glycosidase, and β-N-Acetylglucosaminidase, which are inhibited by SDS. It is imperative to add NP-40 to a final concentration of 1% to your reaction mixture in order to counteract detergent inhibition. Endo D is also inhibited by SDS, however NP-40 does not counteract this inhibition. Finally, common stabilizing reagents such as Tween, Triton X-100, NP-40, octyl glucoside and non-detergent sulfobetaines, as well as traces of organic solvents, can prevent optimal rapid deglycosylation by Rapid PNGase F. Q: What are Glycosidases and their uses? A: Glycosidases are used to get information about the carbohydrate groups attached to glycoproteins and glycopeptides. They come in two varieties, endoglycosidases that cleave entire carbohydrate groups from proteins and exoglycosidases that remove monosaccharides from the non-reduced ends of the carbohydrate. A reduced end is one generated by an endoglycosidase. Researchers frequently use an endoglycosidase followed by one or more exoglycosidases and then analyze the products using SDS-PAGE or various types of liquid chromatography.