Biochemistry

scientificprotocols authored over 3 years ago

Author: Hancock Laboratory Methods

REFERENCE:

Hanock and Benz. 1986. BBA. 860:699-707.

PURPOSE:

Derivitization of amino groups to remove the positive charge characteristics of the protein, for example to determine the importance of amino groups to a protein's function.

MATERIALS:

  1. Purified protein at approximately 0.25mg/ml (diluted in 50mM phosphate buffer pH 6.8, 3mM Na azide, and, for membrane proteins, 0.1% SDS).
  2. Commercial stock solution of acetic anhydride or succinic anhydride.
  3. Microbeaker and flea bar.
  4. pH microprobe to monitor pH during acetylation reaction.
  5. 5N NaOH.

METHOD:

  1. Dilute protein sample to correct concentration as described above.
  2. Using microbeaker and flea bar, set up reaction mix so that it is stirring with the pH probe inserted and pH is monitored. pH must be maintained constantly at between 6.0 and 7.0, or irreversible denaturation of the protein may result. Addition of the acetic anhydride causes a transient drop of pH, which must be corrected with 20µl volumes of 5N NaOH.
  3. Add 2µl acetic anhydride from stock solution every 10 min for 60 min, then let sit for another 60 min at room temp. During this time, maintain pH as described above.
  4. Dialyse vs. 1 litre of 35 mM Na-phosphate buffer pH 6.8, 3mM Na-azide, 0.1% SDS for 4 hrs, then dialyse again in the same overnight.
  5. Residual amino groups can be assayed with trinitro benzene sulphonate (TNBS).

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