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Deglycosylation Kits

Glycosylation often leads to problems in subsequent protein analysis procedures. Glycopeptides generally do not readily ionize during MS analysis leading to insufficient spectral data. Furthermore, proteolytic digestion of the native glycoprotein is often incomplete due to steric hindrance by the oligosaccharides. Removal of the carbohydrate groups from a glycoprotein prior to protein identification is preferred. Compare the features and benefits of our different deglycosylation kits to find the right kit for your application.

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GlycoProfile™ I Enzymatic In-gel N-Deglycosylation Kit
GlycoProfile™ II Enzymatic In-solution N-Deglycosylation Kit
Enzymatic Protein Deglycosylation Kit
Native Protein Deglycosylation Kit
PNGase Fast

GlycoProfile™ I Enzymatic In-gel N-Deglycosylation Kit

Our GlycoProfile™ I Enzymatic In-gel N-Deglycosylation Kit is optimized to provide a convenient and reproducible method to N-deglycosylate and tryptically digest protein samples in 1D or 2D polyacrylamide gel slices for subsequent MS or HPLC analysis. The procedure is suitable for Coomassie™ Blue and Colloidal Coomassie stained gels and may be used with gels silver stained and destained using our ProteoSilver™ Plus Kit (Product No. PROTSIL2). GlycoProfile™ Enzymatic In-gel N-Deglycosylation Kit includes the enzymes and reagents necessary for N-linked deglycosylation and tryptic digestion. The samples can then be desalted and concentrated for analysis by MALDI-TOF or electrospray MS.

Features and Benefits

  • Provides all components for in-gel deglycosylation and trypsinization of protein samples—conveniently prepares deglycosylated protein samples for analysis by MS or HPLC
  • Utilizes PNGase F for the enzymatic removal of N-linked glycans—proteins remain intact, unlike the use of chemical deglycosylation which can degrade the protein
  • Includes Proteomics Grade PNGase F and Trypsin—highly purified enzymes possess no unwanted activities or additives to complicate analysis
  • PNGase F is supplied lyophilized from a low salt buffer—allows reconstitution of the enzyme to any concentration needed
  • Works in solution or with gel slices—allows choice of methods
Components of the GlycoProfile™ I Enzymatic In-gel N-Deglycosylation Kit including PNGase F and trypsin.

Figure 1.Components of GlycoProfile™ I, In-Gel Deglycosylation Kit.

In-Gel Deglycosylation Workflow

Beginning of the In-Gel Deglycosylation workflow starting with excising the protein of interest then placing the gel slice into a siliconized tube.

Step 1

  1. Excise protein of interest
  2. Place gel slice in siliconized tube
    2nd part of the In-Gel Deglycosylation workflow starting with destaining the gel slice and removing the destaining solution and drying the gel in a vacuum concentrator.
    1. Destain gel slice
    2. Remove destaining solution and dry gel in Speed Vac® vacuum concentrator
      3rd part of the In-Gel Deglycosylation workflow starting with rehydrating the gel with PNGase F solution and incubating.
      1. Rehydrate with PNGase F solution
      2. Incubate for 30 minutes at 37 °C
        4th part of the In-Gel Deglycosylation workflow starting with adding water and incubating, then removing the glycan-containing supernatant.
        1. Add water
        2. Incubate for 2 hours to overnight at 37 °C
        3. Remove glycan-containing supernatant, Analyze if desired
          Last step of the In-Gel Deglycosylation workflow starting with washing the gel slice with water, removing liquid, and drying the gel slice.
          1. Wash gel slice with water
          2. Remove and discard liquid
          3. Dry gel slice in Speed Vac® vacuum concentrator
          4. Sample is now ready to be rehydrated in Trypsin solution

            GlycoProfile™ II Enzymatic In-solution N-Deglycosylation Kit

            The GlycoProfile™ Enzymatic In-solution N-Deglycosylation Kit has been optimized to provide a convenient and reproducible method to remove N-linked glycans from glycoproteins and is compatible with subsequent MALDI-TOF mass spectrometric analysis without interference from any of the reaction components. The kit contains sufficient enzyme, glycoprotein standard, and reagents, for a minimum of 20 reactions when the sample size is between one to two mg of a typical glycoprotein.

            Features and Benefits

            • Provides all components for in-solution N-linked deglycosylation of protein samples—conveniently prepares deglycosylated protein samples for analysis by MS, HPLC, and PAGE
            • Reagents are optimized for direct MS analysis—no need for post-reaction sample clean up
            • Utilizes PNGase F for the enzymatic removal of N-linked glycans—proteins remain intact, unlike the use of chemical deglycosylation which can degrade the protein
            • Includes Proteomics Grade PNGase F and Trypsin—highly purified enzymes possess no unwanted activities or additives to complicate analysis
            • PNGase F is supplied lyophilized from a low salt buffer—allows reconstitution of the enzyme to any concentration needed

            In-Solution Deglycosylation Workflow

            Beginning of the In-Solution Deglycosylation workflow starting with aliquoting the glycoprotein sample.

            Step 1

            1. Aliquot glycoprotein sample
              2nd step of the In-Solution Deglycosylation workflow starting with adding denaturant and incubating then cooling to room temp.
              1. Add denaturant
              2. Incubate at 100 °C for 10 minutes
              3. Cool to room temperature
                Last step of the In-Solution Deglycosylation workflow starting with adding reaction buffer and incubating followed by analysis (Mass Spec, PAGE, HPLC).
                1. Add reaction buffer and PNGase F
                2. Incubate at 37 °C for 1 hour
                3. Analyze by MS, PAGE, or HPLC

                  In-solution Deglycosylation

                  Enzymatic Protein Deglycosylation Kit

                  The EDEGLY Kit contains the enzymes and reagents required for deglycosylation of both N- and O-linked glycans.

                  Features and Benefits

                  • Single reaction at neutral pH—no need to adjust conditions for each glycosidase reaction
                  • Native & denaturing procedures—allows flexibility to ensure resulting deglycosylated proteins are compatible with down-stream analysis
                  • Uses highly purified glycosidases, free of contaminating proteases—no degradation of protein results in clear, reliable results
                  • Includes fetuin which has sialylated N- and O-linked oligosaccharides—provides a control to check for efficiency of the deglycosylation
                  • Each reaction deglycosylates 200 µg of glycoprotein—yields enough deglycosylated protein for further analysis
                  • Contains sufficient reagent for the deglycosylation of ~2 mg of protein—allows for the processing of multiple samples

                  Native Protein Deglycosylation Kit

                  The NDEGLY Kit is intended for the deglycoslylation of N-linked oligosaccharides from glycoproteins under native conditions. Particular residues, due to their location in the native protein may be resistant to the traditional degly­cosylation methods using PNGase F. The NDEGLY Kit utilizes endoglycosidases which are less sensitive to protein conformation than PNGase F and, therefore, more suitable for deglycosylation of native proteins.

                  Features and Benefits

                  • Enzymes included are less sensitive to protein conformation than PNGase F—allows the deglycosylation of native proteins
                  • Uses highly purified glycosidases, free of contaminating proteases—no degradation of protein provides reliable, clear data
                  • Each reaction deglycosylates 200 mg of glycoprotein—yields enough deglycosylated protein for further analysis
                  • Contains sufficient reagent for the deglycosylation of ~2 mg of protein—allows for the processing of multiple samples

                  PNGase Fast

                  PNGase Fast allows researchers to confidently and quickly remove N-linked glycosylation from proteins and keep the glycan and protein primary sequence intact. 

                  Features and Benefits

                  • 25 minute prep time
                  • <= 20 minute reaction time
                  • Simple clear procedure using 2 provided components plus 3 common laboratory materials
                  Materials
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