- Email: [email protected]
Elimination of Keratin Contaminant from 2-Mercaptoethanol
98
NOTES & TIPS
proofreading activity, and on crude enzyme preparations obtained during the preparation of DNA polymerase I from Bacillus cereus (results not shown). We did not observe any discrepancies between the novel assay and the conventional assay in either of these cases, although it of course is important that the enzyme preparation should not contain large amounts of double-stranded DNA as a contaminant. In conclusion, we present an uncomplicated, fluorescence-based DNA polymerase assay that utilizes the specific fluorescence properties of the complex between double-stranded DNA and the dye PicoGreen. The assay can be used across polymerase families and is not restricted by elevated assay temperatures or the presence or absence of addition activities, such as exonuclease activities, in the polymerase enzyme under investigation. Calculations of initial velocities (in pmol/ min), based from results from the present assay and a standard curve of double-stranded DNA, give values that correspond to those obtained with a standard radioactivity assay. In addition to being safe and simple, the present assay also is significantly less time-consuming than conventional polymerase assays. REFERENCES 1. Aposhian, H. V., and Kornberg, A. (1962) J. Biol. Chem. 237, 519. 2. Seville, M., West, A. B., Cull, M. G., and McHenry, C. S. (1996) BioTechniques 21, 664, 666, 668, 670, 672. 3. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 4. Ito, J., and Braithwaite, D. K. (1991) Nucleic Acids Res. 19, 4045– 4057. 5. Kong, H., Kucera, R., and Jack, W. (1993) J. Biol. Chem. 268, 1965–1975.
Elimination of Keratin Contaminant from 2-Mercaptoethanol Kalanethee Paul-Pletzer* ,1 and Jerome Parness† ,‡ *Department of Anesthesia, †Department of Pharmacology, and ‡Department of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 Received July 31, 2000
Human epidermal keratin has long been known to be the cause of artifactual, contaminating bands on 1
To whom correspondence should be addressed at Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Research Tower, Room 517, Piscataway, NJ 08854. Fax: (732) 235-4073. E-mail: [email protected]. Analytical Biochemistry 289, 98 –99 (2001) doi:10.1006/abio.2000.4949 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 2 gels, with electrophoretic mobilities corresponding to molecular masses of 50 to 70 kDa (1, 2). Keratin contamination of commercially acquired reducing agents such as 2-mercaptoethanol (2-ME) and dithiothreitol is believed to occur during the manufacturing process (1). These reducing agents are often added to protein electrophoretic sample buffers prior to SDS-PAGE, resulting in keratin contamination of biological samples that may yield confounding and/or misleading results (1). Keratin contamination of protein samples is often encountered as stained bands, spots, and/or vertical streaks on either one- or two-dimensional gels. This can interfere with the analysis of low abundance proteins of overlapping electrophoretic mobilities. Indeed, today, when nanogram amounts of protein are excised from gels for analysis and identification by techniques such as MALDI-mass spectrometry, such contamination becomes unacceptable (3, 4). Furthermore, keratin contamination can be a problem on immunoblots, since polyclonal antibodies raised against various antigens often contain antibodies against this protein (5). Avoidance of direct skin contact with samples, solutions, buffers, and apparatus during gel electrophoresis may reduce but not completely eradicate the problem. Contaminating keratin bands on immunoblots have been eliminated by preadsorption of the anti-sera with keratin, either by directly incubating the anti-sera with keratin or by anti-keratin affinity columns (2, 5). This method is both expensive and time consuming. Here we establish that a simple filtration step is all that is required to eliminate keratin from the reducing agent 2-ME. To demonstrate proof of principle, a fresh bottle of 2-ME (Bio-Rad) was opened in a hood under sterile conditions with the operator wearing latex gloves to reduce the likelihood of in-laboratory contamination with human keratin. All buffers were prepared with the same batch of micropipette tips and the same stock solutions. An aliquot of 2-ME was placed in an Amicon spin column (MWCO 3000) and centrifuged in a microfuge at 13,300 rpm (16,300g) for 30 min at room temperature. The resultant filtrate was used in the preparation of 5X Laemmli sample buffer (6). Two other 5X Laemmli sample buffers were also prepared, one without and one with unfiltered 2-ME. An SDSPAGE gel was run using a 1:5 dilution of these three sample buffers containing no added proteins and transferred onto PVDF membrane using standard buffer 2
Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2-ME, 2-mercaptoethanol; MWCO, molecular weight cutoff; MALDI, matrix-assisted laser desorption ionization.
99
NOTES & TIPS
2. Shapiro, S. Z. (1987) Elimination of the detection of an artifactual 65 kDa keratin band from immunoblots. J. Immuno. Methods 102, 143–146. 3. Parker, K. C., Garrels, J. I., Hines, W., Butler, E. M., McKee, A. H., Patterson, D., and Martin, S. (1998) Identification of yeast proteins from two-dimensional gels: Working out spot cross-contamination. Electrophoresis 19, 1920 –1932. 4. Golaz, O., Wilkins, M. R., Sanchez, J. C., Appel, R. D., Hochstrasser, D. F., and Williams, K. L. (1996) Identification of proteins by their amino acid composition: An evaluation of the method. Electrophoresis 17, 573–579. 5. Girault, J. A., Gorelick, F. S., and Greengard, P. (1989) Improving the quality of immunoblots by chromatography of polyclonal antisera on keratin affinity columns. Anal. Biochem. 182, 193–196. 6. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685.
FIG. 1. Keratin contamination of 2-ME eliminated by membrane filtration. Laemmli sample buffer (1X) prepared in the absence of 2-ME (lane 1), or the presence of filtered (lane 2) or unfiltered (lane 3) 2-ME (2.88 mM final concentration), as described in the text, was loaded and run on a 7.5% SDS-PAGE gel. The contents of the gel were electroblotted onto a PVDF membrane under standard conditions and the presence of keratin on the membrane was determined by immunoblotting first with a guinea pig anti-keratin antibody (delipidized whole serum, Sigma) and secondarily with an alkaline phosphatase-conjugated goat anti-guinea pig antibody (Sigma). Color was then developed with 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride using standard protocols.
3-Hydroxybenzoic Acid as an Internal Standard for the High-Pressure Liquid Chromatography Quantitation of Salicylic Acid in Plants 1 Steven M. Eshita 2 USDA Forest Service, Northeastern Research Station, 359 Main Road, Delaware, OH 43015 Received October 12, 2000
conditions. The membrane was then probed with guinea pig anti-keratin, and the immunoreactive bands were visualized with alkaline phosphatase-conjugated, goat anti-guinea pig antibody using standard protocols. The results, presented in Fig. 1, clearly demonstrate that keratin is present only in the lane in which the Laemmli sample buffer was prepared with unfiltered 2-ME (lane 3). No keratin was found in the lanes containing Laemmli sample buffer prepared with filtered 2-ME (lane 2) or prepared in its absence (lane 1). Hence, the source of keratin is 2-ME and this contaminant can be eliminated by passing the reducing agent through an inert filter with a nominal 3000 molecular weight cutoff. We hope that this simple and effective means of solving the problem of keratin contamination in gel electrophoresis will be welcomed by both biochemists and manufacturers of reducing agents for the life sciences community. Acknowledgments. This work was supported by Grant 5 RO1 AR45593-02 from NIH-NIAMS, and by clinical funds of the Department of Anesthesia, UMDNJ-Robert Wood Johnson Medical School. We are grateful to Kenneth E. Clune for his suggestions.
REFERENCES 1. Ochs, D. (1983) Protein contaminants of sodium dodecyl sulfatepolyacrylamide gels. Anal. Biochem. 135, 470 – 474.
Salicylic acid (2-hydroxybenzoic acid; SA) 3 is an important molecule in plant defense responses to pathogens and stress (1). Research on defense signal transduction in plants may require quantitation of SA and related aromatic secondary metabolites. These molecules cannot be analyzed directly in plant tissue but are extractable and quantified routinely by high-performance liquid chromatography (HPLC), requiring internal standardization for recovery correction. Selection criteria for an internal standard include: (a) specific detection (lack of interference by other compounds), (b) endogenous absence in sample, (c) high detection sensitivity (requiring lower amounts of internal standard), and (d) comparable recovery to SA. Radiolabeled SA meets these criteria, but radioisotope can be expensive and troublesome to handle 1
The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture or the Forest Service of any product or service to the exclusion of others that may be suitable. 2 Fax: (740) 368-0152. E-mail: [email protected]. 3 Abbreviations used: SA, salicylic acid; HPLC, high-pressure liquid chromatography; 3HBA, 3-hydroxybenzoic acid; UV, ultraviolet; 4HBA, 4-hydroxybenzoic acid; 4HCA, para-coumaric acid; BA, benzoic acid; 2HCA, ortho-coumaric acid; CA, trans-cinnamic acid; A 236, absorbance at 236 nm; ANOVA, analysis of variance; FW, fresh weight. Analytical Biochemistry 289, 99 –102 (2001) doi:10.1006/abio.2000.4962 0003-2697/01 $35.00
NOTES & TIPS
proofreading activity, and on crude enzyme preparations obtained during the preparation of DNA polymerase I from Bacillus cereus (results not shown). We did not observe any discrepancies between the novel assay and the conventional assay in either of these cases, although it of course is important that the enzyme preparation should not contain large amounts of double-stranded DNA as a contaminant. In conclusion, we present an uncomplicated, fluorescence-based DNA polymerase assay that utilizes the specific fluorescence properties of the complex between double-stranded DNA and the dye PicoGreen. The assay can be used across polymerase families and is not restricted by elevated assay temperatures or the presence or absence of addition activities, such as exonuclease activities, in the polymerase enzyme under investigation. Calculations of initial velocities (in pmol/ min), based from results from the present assay and a standard curve of double-stranded DNA, give values that correspond to those obtained with a standard radioactivity assay. In addition to being safe and simple, the present assay also is significantly less time-consuming than conventional polymerase assays. REFERENCES 1. Aposhian, H. V., and Kornberg, A. (1962) J. Biol. Chem. 237, 519. 2. Seville, M., West, A. B., Cull, M. G., and McHenry, C. S. (1996) BioTechniques 21, 664, 666, 668, 670, 672. 3. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 4. Ito, J., and Braithwaite, D. K. (1991) Nucleic Acids Res. 19, 4045– 4057. 5. Kong, H., Kucera, R., and Jack, W. (1993) J. Biol. Chem. 268, 1965–1975.
Elimination of Keratin Contaminant from 2-Mercaptoethanol Kalanethee Paul-Pletzer* ,1 and Jerome Parness† ,‡ *Department of Anesthesia, †Department of Pharmacology, and ‡Department of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 Received July 31, 2000
Human epidermal keratin has long been known to be the cause of artifactual, contaminating bands on 1
To whom correspondence should be addressed at Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Research Tower, Room 517, Piscataway, NJ 08854. Fax: (732) 235-4073. E-mail: [email protected]. Analytical Biochemistry 289, 98 –99 (2001) doi:10.1006/abio.2000.4949 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 2 gels, with electrophoretic mobilities corresponding to molecular masses of 50 to 70 kDa (1, 2). Keratin contamination of commercially acquired reducing agents such as 2-mercaptoethanol (2-ME) and dithiothreitol is believed to occur during the manufacturing process (1). These reducing agents are often added to protein electrophoretic sample buffers prior to SDS-PAGE, resulting in keratin contamination of biological samples that may yield confounding and/or misleading results (1). Keratin contamination of protein samples is often encountered as stained bands, spots, and/or vertical streaks on either one- or two-dimensional gels. This can interfere with the analysis of low abundance proteins of overlapping electrophoretic mobilities. Indeed, today, when nanogram amounts of protein are excised from gels for analysis and identification by techniques such as MALDI-mass spectrometry, such contamination becomes unacceptable (3, 4). Furthermore, keratin contamination can be a problem on immunoblots, since polyclonal antibodies raised against various antigens often contain antibodies against this protein (5). Avoidance of direct skin contact with samples, solutions, buffers, and apparatus during gel electrophoresis may reduce but not completely eradicate the problem. Contaminating keratin bands on immunoblots have been eliminated by preadsorption of the anti-sera with keratin, either by directly incubating the anti-sera with keratin or by anti-keratin affinity columns (2, 5). This method is both expensive and time consuming. Here we establish that a simple filtration step is all that is required to eliminate keratin from the reducing agent 2-ME. To demonstrate proof of principle, a fresh bottle of 2-ME (Bio-Rad) was opened in a hood under sterile conditions with the operator wearing latex gloves to reduce the likelihood of in-laboratory contamination with human keratin. All buffers were prepared with the same batch of micropipette tips and the same stock solutions. An aliquot of 2-ME was placed in an Amicon spin column (MWCO 3000) and centrifuged in a microfuge at 13,300 rpm (16,300g) for 30 min at room temperature. The resultant filtrate was used in the preparation of 5X Laemmli sample buffer (6). Two other 5X Laemmli sample buffers were also prepared, one without and one with unfiltered 2-ME. An SDSPAGE gel was run using a 1:5 dilution of these three sample buffers containing no added proteins and transferred onto PVDF membrane using standard buffer 2
Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2-ME, 2-mercaptoethanol; MWCO, molecular weight cutoff; MALDI, matrix-assisted laser desorption ionization.
99
NOTES & TIPS
2. Shapiro, S. Z. (1987) Elimination of the detection of an artifactual 65 kDa keratin band from immunoblots. J. Immuno. Methods 102, 143–146. 3. Parker, K. C., Garrels, J. I., Hines, W., Butler, E. M., McKee, A. H., Patterson, D., and Martin, S. (1998) Identification of yeast proteins from two-dimensional gels: Working out spot cross-contamination. Electrophoresis 19, 1920 –1932. 4. Golaz, O., Wilkins, M. R., Sanchez, J. C., Appel, R. D., Hochstrasser, D. F., and Williams, K. L. (1996) Identification of proteins by their amino acid composition: An evaluation of the method. Electrophoresis 17, 573–579. 5. Girault, J. A., Gorelick, F. S., and Greengard, P. (1989) Improving the quality of immunoblots by chromatography of polyclonal antisera on keratin affinity columns. Anal. Biochem. 182, 193–196. 6. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685.
FIG. 1. Keratin contamination of 2-ME eliminated by membrane filtration. Laemmli sample buffer (1X) prepared in the absence of 2-ME (lane 1), or the presence of filtered (lane 2) or unfiltered (lane 3) 2-ME (2.88 mM final concentration), as described in the text, was loaded and run on a 7.5% SDS-PAGE gel. The contents of the gel were electroblotted onto a PVDF membrane under standard conditions and the presence of keratin on the membrane was determined by immunoblotting first with a guinea pig anti-keratin antibody (delipidized whole serum, Sigma) and secondarily with an alkaline phosphatase-conjugated goat anti-guinea pig antibody (Sigma). Color was then developed with 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride using standard protocols.
3-Hydroxybenzoic Acid as an Internal Standard for the High-Pressure Liquid Chromatography Quantitation of Salicylic Acid in Plants 1 Steven M. Eshita 2 USDA Forest Service, Northeastern Research Station, 359 Main Road, Delaware, OH 43015 Received October 12, 2000
conditions. The membrane was then probed with guinea pig anti-keratin, and the immunoreactive bands were visualized with alkaline phosphatase-conjugated, goat anti-guinea pig antibody using standard protocols. The results, presented in Fig. 1, clearly demonstrate that keratin is present only in the lane in which the Laemmli sample buffer was prepared with unfiltered 2-ME (lane 3). No keratin was found in the lanes containing Laemmli sample buffer prepared with filtered 2-ME (lane 2) or prepared in its absence (lane 1). Hence, the source of keratin is 2-ME and this contaminant can be eliminated by passing the reducing agent through an inert filter with a nominal 3000 molecular weight cutoff. We hope that this simple and effective means of solving the problem of keratin contamination in gel electrophoresis will be welcomed by both biochemists and manufacturers of reducing agents for the life sciences community. Acknowledgments. This work was supported by Grant 5 RO1 AR45593-02 from NIH-NIAMS, and by clinical funds of the Department of Anesthesia, UMDNJ-Robert Wood Johnson Medical School. We are grateful to Kenneth E. Clune for his suggestions.
REFERENCES 1. Ochs, D. (1983) Protein contaminants of sodium dodecyl sulfatepolyacrylamide gels. Anal. Biochem. 135, 470 – 474.
Salicylic acid (2-hydroxybenzoic acid; SA) 3 is an important molecule in plant defense responses to pathogens and stress (1). Research on defense signal transduction in plants may require quantitation of SA and related aromatic secondary metabolites. These molecules cannot be analyzed directly in plant tissue but are extractable and quantified routinely by high-performance liquid chromatography (HPLC), requiring internal standardization for recovery correction. Selection criteria for an internal standard include: (a) specific detection (lack of interference by other compounds), (b) endogenous absence in sample, (c) high detection sensitivity (requiring lower amounts of internal standard), and (d) comparable recovery to SA. Radiolabeled SA meets these criteria, but radioisotope can be expensive and troublesome to handle 1
The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture or the Forest Service of any product or service to the exclusion of others that may be suitable. 2 Fax: (740) 368-0152. E-mail: [email protected]. 3 Abbreviations used: SA, salicylic acid; HPLC, high-pressure liquid chromatography; 3HBA, 3-hydroxybenzoic acid; UV, ultraviolet; 4HBA, 4-hydroxybenzoic acid; 4HCA, para-coumaric acid; BA, benzoic acid; 2HCA, ortho-coumaric acid; CA, trans-cinnamic acid; A 236, absorbance at 236 nm; ANOVA, analysis of variance; FW, fresh weight. Analytical Biochemistry 289, 99 –102 (2001) doi:10.1006/abio.2000.4962 0003-2697/01 $35.00