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Notice of Adoption of Harmonized Standard

Targeted Official Date:

01–Dec–2015

Expert Committee:

General Chapters—Biological Analysis

Coordinating Pharmacopoeia:

European Pharmacopoeia

A revision to the harmonized standard for Polyacrylamide Gel Electrophoresis has been approved by the Pharmacopeial Discussion Group (PDG) as described in its PDG Stage 6 Rev. 1 Sign-Off Cover Page. Having reached Stage 6 of the PDG process, the Polyacrylamide Gel Electrophoresis General Chapter has been formally approved by the General Chapters—Biological Analysis Expert Committee in accordance with the Rules and Procedures of the 2010–2015 Council of Experts.

Changes from the existing USP–NF General Chapter include:

Prior to the Introduction, Removed the harmonization preamble and notes. In the Introduction, The national text section called “General Principles of Electrophoresis” was removed from the General Chapter since the important principles are sufficiently covered in the harmonized text. In the section Denaturing Polyacrylamide Gel Electrophoresis, Updated the text to provide guidance for measurement of molecules below 14, 000 Da, including the use of gradient gels and substitution of glycine with tricine as the trailing ion in the electrophoresis running buffer. The section Nonreducing Conditions Contains additional guidance regarding the impact of dissociation and appropriate determination of molecular mass determinations. In the section Preparing Vertical Discontinuous Buffer SDS-Polyacrylamide Gels An introductory paragraph was added clarifying that commercially prepared gels and reagents can be used and that in this case manufacturers’ procedures must be followed. This paragraph also states that pretreatment of prepared solutions may be necessary to remove impurities. The subsection “Plate Preparation” was renamed “Assembling the Gel-Molding Cassette” for accuracy. Slight updates for current best practices were made to this section (e.g., recommendation of an alcohol rinse and air drying the plates after washing rather than drying with a paper towel or tissue). The sections Preparation of the Sample and Mounting the Gel in the Electrophoresis Apparatus and Electrophoretic Separation Contain the text of the previously named Electrophoretic Separation with updates for best practices. This section also clarifies that if a monograph describes a different sample preparation then the user should follow the monograph guidance, not the chapter guidance. A new section Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis- Gradient Gels was added containing additional guidance for these types of gels. In the section Detection of Proteins in Gels, Text was expanded to discuss advantages and disadvantages of common staining methods and to also add other options such as fluorescent stains The section Drying of Gels was renamed Recording of Results and this section was updated for current best practices, including allowing recording of results before gel drying. In the section Validation of the Test, The text was updated to explain appropriate gel separation and use of results demonstrating a sigmoidal data plot. This section also clarifies that sensitivity must be validated and this can be accomplished with a suitable reference control protein. The section Quantification of Impurities was slightly edited for greater clarity.
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