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CHAPTER

Electrophoresis

CONTENTS Separation Ranges of Polyacrylamide Gels Preparation of Polyacrylamide Gels Buffer Mixtures Commonly Used for Polyacrylamide Gel Electrophoresis Proteins for Internal Standardization of Polyacrylamide Gel Electrophoresis Chromogenic Stains for Gels Fluorescent Stains for Gels

Copyright 2003 CRC Press, LLC

SEPARATION RANGES OF POLYACRYLAMIDE GELS The following table provides a rough guide to the separation ranges of polyacrylamide gels that have varying gel concentrations, T, expressed in percent, as a function of relative molecular mass.1

REFERENCES
1. Andrews, A.T., Electrophoresis: Theory Techniques and Biochemical and Clinical Applications, 2nd ed., Oxford University Press, Oxford, 1986.
Separation Ranges of Polyacrylamide Gels T (Percent) 35 512 1015 15+ Optimum Relative Molecular Mass Range Above 100,000 20,000150,000 10,00080,000 Below 15,000

From Andrews, A.T., Electrophoresis: Theory Techniques and Biochemical and Clinical Applications, 2nd ed., Oxford University Press, Oxford, 1986. With permission.

Copyright 2003 CRC Press, LLC

PREPARATION OF POLYACRYLAMIDE GELS The following table provides in recipe format the typical proportions of reagents needed to prepare 100 ml of the starting material for polyacrylamide gels.1 The factor T is the gel concentration and is related to the ability to separate a given relative molecular mass range. Typically, the tertiary aliphatic amines N,N,N,N-tetramethylethylenediamine (TEMED) or 3-dimethylaminopropionitrile (DMAPN) are used to catalyze the reaction. Note that gelation does not occur readily below T = 2.5%.

REFERENCES
1. Andrews, A.T., Electrophoresis: Theory Techniques and Biochemical and Clinical Applications, 2nd ed., Oxford University Press, Oxford, 1986.

Preparation of Polyacrylamide Gels Amounts Required for Gels With Constituent Acrylamide Biscrylamide TEMED or DMAPN Ammonium persulfate T = 5% 4.75 0.25 0.05 0.05 g g ml g T = 7.5% 7.125 g 0.375 g 0.05 ml 0.05 g T = 10% 9.50 0.50 0.05 0.05 g g ml g

From Andrews, A.T., Electrophoresis: Theory Techniques and Biochemical and Clinical Applications, 2nd ed., Oxford University Press, Oxford, 1986. With permission.

Copyright 2003 CRC Press, LLC

BUFFER MIXTURES COMMONLY USED FOR POLYACRYLAMIDE GEL ELECTROPHORESIS The following table provides suggested buffers used for polyacrylamide gel electrophoresis. This list is by no means exhaustive; however, these buffers are the most common.1

REFERENCES
1. Andrews, A.T., Electrophoresis: Theory Techniques and Biochemical and Clinical Applications, 2nd ed., Oxford University Press, Oxford, 1986.
Buffer Mixtures Commonly Used for Polyacrylamide Gel Electrophoresis Approximate pH Range 2.46.0 2.83.8 4.05.5 5.27.0 6.08.0 7.08.5 7.29.0 8.510.0 9.010.5 9.011.0 Primary Buffer Constituent 0.1 M citric acid 0.05 M formic acid 0.05 M acetic acid 0.05 M maleic acid 0.05 M KH2PO4 or NaH2PO4 0.05 M Sodium diethyl-barbiturate (veronal) 0.05 M Tris 0.015 M Na2B4O7 0.05 M glycine 0.025 M NaHCO3 pH Adjusted to the Desired Value With 1 1 1 1 1 1 1 1 1 1 M M M M M M M M M M NaOH NaOH NaOH or tris NaOH or tris NaOH HCl HCl or glycine HCl or NaOH NaOH NaOH

From Andrews, A.T., Electrophoresis: Theory Techniques and Biochemical and Clinical Applications, 2nd ed., Oxford University Press, Oxford, 1986. With permission.

Copyright 2003 CRC Press, LLC

PROTEINS FOR INTERNAL STANDARDIZATION OF POLYACRYLAMIDE GEL ELECTROPHORESIS The following table provides a list of proteins that may be used as internal standards, along with their isoelectric points, pI, in quantitative applications of polyacrylamide gel electrophoresis. These proteins may be used in isoelectric focusing or in SDS-PAGE. The isoelectric points are reported at 25C.1

REFERENCES
1. Andrews, A.T., Electrophoresis: Theory Techniques and Biochemical and Clinical Applications, 2nd ed., Oxford University Press, Oxford, 1986.

Proteins for Internal Standardization of Polyacrylamide Gel Electrophoresis Isoelectric Point (pI at 25C) 10.0 9.3 9.0 8.9 8.2 7.3 6.5 5.7 5.3 5.1 5.1 4.7 4.4 4.3 Relative Molecular Mass 14,000 12,256 23,600 13,500 17,500 17,500 130,000 11,466 36,552 36,724 67,000 45,000 140,000 14,146

Protein Lysozyme Cytochrome C (horse) Chymotrypsinogen A (ox) Ribonuclease A Myoglobin (sperm whale) Myoglobin (horse) Erythroagglutinin (red kidney bean) Insulin (beef) -Lactoglobulin B -Lactoglobulin A Bovine serum albumin Ovalbumin Alkaline phosphatase (calf intestine) -Lactalbumin

From Andrews, A.T., Electrophoresis: Theory Techniques and Biochemical and Clinical Applications, 2nd ed., Oxford University Press, Oxford, 1986. With permission.

Copyright 2003 CRC Press, LLC

CHROMOGENIC STAINS FOR GELS The following table provides common stain reagents for use in electrophoresis gels.1

REFERENCES
1. Melvin, M., Electrophoresis (Analytical Chemistry by Open Learning), John Wiley & Sons, Chichester, 1987.

Chromogenic Stains for Gels Types of Substance Stained Amino acids, peptides, and proteins Proteins Staining Reagent Ninhydrin Comments Very sensitive stain for amino acids, either free or combined in polypeptides; used after paper electrophoresis Binds to cationic groups on proteins; adsorbs onto cellulose, giving high background staining with paper and dehydration and shrinkage of polyacrylamide gels Binds to basic groups on proteins and also by nonpolar interactions; widely used stain Used routinely in clinical laboratories for cellulose acetate and starch gels; very rapid staining reaction that leaves a clear background Stains the sugar moiety Specically indicates the presence of copper Stained product can be assessed quantitatively

Amido Black 10B

Coomassie Brilliant Blue Ponceau S (Ponceau Red) Glycoproteins Copper-containing proteins Polynucleotides, including RNA and DNA Alcian Blue Alizarin Blue S Acridine orange

Pyronine Y (or G)

Gives a permanent staining, so electrophoretogram can be stored for several weeks

Proteins, lipids, carbohydrates, polynucleotides

StainsAll

Wide applicability, as it forms characteristic colored products with many different types of molecule; low sensitivity

From Melvin, M., Electrophoresis (Analytical Chemistry by Open Learning), John Wiley & Sons, Chichester, 1987. With permission.

Copyright 2003 CRC Press, LLC

FLUORESCENT STAINS FOR GELS The following table provides common uorescent stain reagents for use in electrophoresis.1 Note that these agents are typically applied in small amounts before electrophoresis. Other stains are available as proprietary materials; consult reviews on staining procedures for additional materials.2,3

REFERENCES
1. Melvin, M., Electrophoresis (Analytical Chemistry by Open Learning), John Wiley & Sons, Chichester, 1987. 2. Williams, L.R., Staining nucleic acids and proteins in electrophoresis gels, Biotech. Histochem., 76, 127, 2001. 3. Allen, R. and Budowle, B., Protein Staining and Identication Techniques, BioTechniques Press, Westborough, MA, 1999.

Fluorescent Stains for Gels Types of Substance Stained Proteins Staining Reagent Dansyl chloride 1-Anilino-8-naphthalene sulphonic acid Fluorescamine Acridine orange Ethidium bromide Comments Reacts with amine groups Nonuorescent, but gives uorescent product Nonuorescent, but gives uorescent product

Polynucleotides, including RNA and DNA Double-stranded polynucleotides

Very sensitive; widely used with agarose gels

From Melvin, M., Electrophoresis (Analytical Chemistry by Open Learning), John Wiley & Sons, Chichester, 1987. With permission.

Copyright 2003 CRC Press, LLC