O-Glycosidase

Product Information

 
O-Glycosidase, also known as Endo-α-N-Acetylgalactosaminidase, catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins.

Substrate Specificity:

Product Source

Cloned from Enterococcus faecalis and expressed in E. coli (1).

This product is related to the following categories:

Endoglycosidases Products,

Proteome Analysis Products

This product can be used in the following applications:

Expression Systems,

Glycan Sequencing,

Protein Digestion,

Recombinant Glycoprotein Expression,

Glycoprotein Analysis

• Reagents Supplied

  The following reagents are supplied with this product:

  NEB https://www.neb.com/en/#	Component Name	Component https://www.neb.com/en/#	Stored at (°C)	Amount	Concentration
  P0733S     -20       O-Glycosidase P0733SVIAL -20 1 x 0.05 ml 40,000,000 units/ml   GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X   Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X   NP-40 B2704SVIAL -20 1 x 1 ml 10 %
  P0733L     -20       O-Glycosidase P0733LVIAL -20 1 x 0.25 ml 40,000,000 units/ml   GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X   Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X   NP-40 B2704SVIAL -20 1 x 1 ml 10 %

• Properties & Usage

  Unit Definition

  One unit is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of neuraminidase-digested, non-denatured fetuin (2) in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).
  
  1X Glycoprotein Denaturing Buffer
  0.5% SDS
  40 mM DTT
  
  1X NP-40
  1% NP-40 in MilliQ-H₂O

  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
  1 mM EDTA
  pH 7.5 @ 25°C

  Heat Inactivation

  65°C for 10 minutes

  Molecular Weight

  Apparent: 147 kDa
  

  Unit Assay Conditions

  Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of neuraminidase digested fetuin with 1X GlycoBuffer 2. The reaction mix is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by the Morgan and Elson Assay (1).

• Advantages and Features

  Application Features

  • Removal of Core 1 and Core 3 O-linked disaccharide glycans from glycoproteins

• Related Products

  Companion Products

  • O-Glycosidase & Neuraminidase Bundle
  • α2-3,6,8 Neuraminidase
  • PNGase F
  • PNGase F (Glycerol-free)
  • Endo H_f
  • Endo H

• Product Notes

  1. Since O-Glycosidase is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present time. Double digest with Endo H must have NP-40 present (NP-40 does not inhibit Endo H).
  2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
  3. It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides. A general Neuraminidase (https://www.neb.com/en/#P0720) or the O-Glycosidase and Neuraminidase bundle (https://www.neb.com/en/#E0540S)  is recommended.
  4. Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.

• References

  1. Morgan, W.T.J. and Elson, L.A. (1994). Biochem. J.. 28, 988-995.

Protocols, Manuals & Usage

• Protocols

  1. O-Glycosidase Application Note 1 (P0733)
  2. O-Glycosidase (P0733)
  3. Endo-α-N-Acetylgalactosaminidase Application Note 1

• Application Notes

  • AppNote_Enzymatic_Deglycosylation_of_Protein_Containing_Core_1_O-glycans_with_O-Glycosidase
  • Analysis of a Fusion Protein using the Protein Deglycosylation Mix II and Mass Spectrometry
  • Enzymatic Deglycosylation of a Protein Containing Core 1 O-glycans with O-Glycosidase

Tools & Resources

• Selection Charts

  • Endoglycosidase Selection Chart

FAQs & Troubleshooting

• FAQs

  1. What are the typical reaction conditions for O-Glycosidase?
  2. I tried using O-Glycosidase on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
  3. How much O-Glycosidase should I use to remove my carbohydrate under native conditions?
  4. Do detergents inhibit O-Glycosidase?
  5. Can Neuraminidase be used together in a digest with PNGase F and O-Glycosidase?
  6. Can I double digest PNGase F and O-Glycosidase?
  7. What is the difference between PNGase F, Endo H and O-Glycosidase?
  8. 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?
  9. What are Glycosidases and their uses?
  10. Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase?
  11. How do I inhibit O-Glycosidase?

• Tech Tips

  Don’t forget to include Neuraminidase (P0720) in your reaction! It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides.

  NEB’s O-Glycosidase is the only available O-Glycosidase that catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins

  You can use this enzyme under native or denaturing conditions

  Under native conditions we recommend adding more enzyme and using longer incubation times

  Under denaturing conditions the enzyme activity is increased two-fold. However, this observation may be substrate dependent.

Citations & Technical Literature

• Citations

  Product Citation Tool

• Feature Articles

  • Using Glycosidases to Remove Trim or Modify Glycans on Therapeutic Protein
  • O-glycan analysis of therapeutic proteins enabled by O-glycoprotease

Quality, Safety & Legal

• Quality Control Assays

  Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

• Specifications & Change Notifications

  The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

  • P0733S_L_v1

• Certificate of Analysis

  The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

  • P0733S_L_v1_0031603
  • P0733S_L_v1_0031612
  • P0733S_L_v1_0031706
  • P0733S_L_v1_0031801
  • P0733S_v1_10011712
  • P0733S_v1_10015197
  • P0733L_v1_10025560
  • P0733S_v1_10027227
  • P0733S_v1_10033432
  • P0733S_v1_10039238
  • P0733L_v1_10043512
  • P0733S_v1_10043510
  • P0733L_v1_10050882
  • P0733S_v1_10051572
  • P0733S_v1_10054778
  • P0733S_v1_10057983
  • P0733S_v1_10062547
  • P0733S_v1_10077195
  • P0733L_v1_10082483
  • P0733L_v1_10097236
  • P0733S_v1_10084150
  • P0733L_v1_10106703
  • P0733S_v1_10112430
  • P0733L_v1_10128264
  • P0733S_v1_10126416
  • P0733S_v1_10149065
  • P0733L_v1_10149063
  • P0733S_v1_10175375
  • P0733L_v1_10176604
  • P0733S_v1_10195129
  • P0733L_v1_10212858
  • P0733S_v1_10226279
  • P0733L_v1_10245827
  • P0733L_v1_10257232
  • P0733S_v1_10260303

• Safety Data Sheets

  The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

  • O-Glycosidase
  • GlycoBuffer 2
  • Glycoprotein Denaturing Buffer
  • NP-40

• Legal and Disclaimers

  Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
  
  This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
  
  New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.