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Sugar detection by Enzyme Treatment and Electrophoresis


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   Tools for sugar chain research!!
• Lectins
• GlyScope Series

It is possible to identify the presence of a sugar chains, by cutting off the sugar chain using enzymes and examining whether the molecular weight of the protein decreases or not. Here, by using endo-acting enzymes, N-glycanase and O-glycanase, the molecular weight changes during enzyme processing and can be examined by SDS-PAGE.

1. Equipment and apparatus.

Thermostat
Electrophoresis system for SDS-PAGE

2. N-glycanase reaction.

2-1. Reagents

* 0.2M sodium phosphate buffer solution (pH8.6)
* Sodium Dodecylsulfate, SDS
* β-Mercaptoethanol
* Nonidet P-40iNP-40j(Surface-active agent)
* N-glycanase (N-glycosidaseF) <from flavobacterium meningosepticum>

2-2. Procedures.

  1. Add 100ul 0.5%SDS, 1% β-Mercaptoethanol / 0.2M sodium phosphate buffer solution to glycoprotein (100ug), soak in a thermostat (90-100°C) for 3 minutes.
  2. In the meantime, prepare a 10ul tube.
  3. Add 5ul 7.5%NP-40 or distilled water.
  4. Add 5ul N-glycanase, make the final enzyme concentration to be 10~30U/ml.
  5. React for 18 hours at 37°C.
  6. Apply to SDS-PAGE.

3. O-glycanase reaction.

3-1. Reagents

*0.2M sodium phosphate buffer solution (pH6.0)
*O-glycanase (endo-α-N-acetylgalactosaminidase) <from Streptococcus or Diplococcus pneumoniae>

3-2. Procedures.

  1. Add 200ul 0.2M sodium phosphate buffer solution (pH6.0) to glycoprotein (25~35ug), mix well.
  2. Add 3mU O-glycanase.
  3. React for 16 hours at 37°C.
  4. Apply to SDS-PAGE.

* In the case of O-glycanase, please note that substrate specificity is different according to the origin of microorganism species; not all O-linked sugar chains are cut off. For the frequently used Diplococcus pneumonia (Streptococcus pneumonia), only Galβ1→3GalNAc→Ser(Thr) disaccharide units (type 1) were extricated.

4. An analysis example

After SDS-PAGE, stained by CBB was carried out(Coomassie Brilliant Blue) (schema)

Lane 1 sample (protein)
Lane 2 sample processed by N-glycanase
Lane 3 sample processed by O-glycanase

If results are obtained like above, it is presumed that there are N-linked sugar chains and no O-linked sugar chains (type 1) in the sample.

5. Judgement points.

According to the results obtained after electrophoresis, if the band positions of the enzyme are identicle and the bands obtained from samples not processed are different (when the difference is small), it can be considered that the protein contains sugar chains.

 

 

   Tools for sugar chain research!!
• Lectins
• GlyScope Series

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


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