Shreffler:Periodate Treatment

Overview

Periodic acid can be used to degrade carbohydrate structures in order to test for the role of glycans in biological assays.

Materials

• 15 mL polypropylene tubes
• PBS
• 50 mM Sodium Acetate Buffer, pH 4.5
• 100 mM Sodium Borate Buffer, pH 8.0
• Sodium Metaperiodate: Sigma cat no. S1878
• Glycerol
• Sodium Borohydride: Sigma cat no. 480886

Procedure

Treatment of soluble protein:

1. Bring CPE to ~3 mg/mL in 1 mL PBS
2. Add 1 ml 100 mM sodium metaperiodate in PBS (pH 7.2)
3. Incubate at 4°C in dark for 18h (O/N)
4. Add 1 ml 260 mM glycerol
5. Dialyze against PBS
6. Protein assay and gel (approx protein conc 1 mg/mL

Treatment of plate bound protein (from reference 2):

1. Coat wells with CPE per routine
2. Add 0.25 mM sodium acetate buffer, pH 4.5 with 10 mM sodium metaperiodate
3. Incubate 2h at 37°C in the dark
4. Wash 1X with dH₂O
5. Add 0.5M sodium borate buffer, pH 8.0, containing 0.5 M Sodium borohydride for 30 min at 25°C
6. Use immediately

Discussion

discuss this protocol

References

1. Sato C, Inoue S, Matsuda T, and Kitajima K. Development of a highly sensitive chemical method for detecting alpha2-->8-linked oligo/polysialic acid residues in glycoproteins blotted on the membrane. Anal Biochem. 1998 Aug 1;261(2):191-7. DOI:10.1006/abio.1998.2718 |
2. Hino S, Matsubara T, Urisu A, Aoki N, Sato C, Okajima T, Nadano D, and Matsuda T. Periodate-resistant carbohydrate epitopes recognized by IgG and IgE antibodies from some of the immunized mice and patients with allergy. Biochem Biophys Res Commun. 2009 Mar 13;380(3):632-7. DOI:10.1016/j.bbrc.2009.01.138 |

Contact

• Who has experience with this protocol?
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