Research Reports
Column-based method to simultaneously extract DNA, RNA, and proteins from the same sample Jorge M. Tolosa1,2, John E. Schjenken1, Theodora D. Civiti1, Vicki L. Clifton1, and Roger Smith1 BioTechniques 43:799-804 (December 2007) doi 10.2144/000112594
We describe a procedure for the simultaneous extraction of proteins and nucleic acids from the same experimental sample allowing for direct correlations between genetic, genomic, and proteomic data. This approach, using commercially available column-based nucleic acid extraction kits, requires no hazardous chemicals and is a quick, reliable, and consistent method for concomitant protein extraction. Buffer choice is critical to completely solubilize all proteins in the sample. Proteins solubilized in radioimmunoprecipitation assay (RIPA) buffer did not represent the entire profile when compared with conventionally extracted proteins using the same buffer at the one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) level, however, proteins extracted from the columns and solubilized in a two-dimensional (2-D) electrophoresis lysis buffer showed a similar profile to conventionally extracted proteins when analyzed at both the 1-D and the 2-D level. We further showed that proteins extracted using these methods were compatible with Western blot analysis. This technique provides a simple and effective way to analyze protein and nucleic acids simultaneously from the same sample without affecting yield and quality.
INTRODUCTION Biological complexity emerges from different organizational levels in a highly regulated space-time coordination of processes that involves the participation and orchestrated interaction of DNA, RNA, and proteins between each other and the environment. Fully understanding normal biological processes such as cell differentiation, development and aging, and pathological conditions requires integrated genomic, transcriptional, and proteomic studies (1–3), which demand the simultaneous isolation of DNA, RNA, and proteins from the same sample. Quick and reliable methods that perform simultaneous extraction of DNA, RNA, and proteins from a single sample are ideal for the generation of matched samples that can save time and money and allow for the efficient use of small and precious biological samples. Researchers are increasingly turning away from classic RNA and protein extraction techniques, such as phenol-chloroform separation (4) or time-consuming cesium chloride gradient centrifugation, because of 1University
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the hazardous chemicals used and that the methods are generally unsuited for routine use in the laboratory. Spin column technology is a simple and quick approach to extracting nucleic acids from small biological samples. Furthermore, most column-based procedures do not require the amount of hazardous chemicals that are used in traditional nucleic acid extraction procedures (5). Recently, Morse and coworkers (5) discussed the combined extraction of RNA and proteins using RNA spin column–based technology, and Hummon et al. (6) showed an improved method for isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from the same sample. However, none of these authors did a complete analysis of the proteins obtained at the level of two-dimensional (2-D) electrophoresis to compare the protein profile obtained with conventional methods used in proteomics studies. Here we present a methodology to simultaneously extract RNA/proteins and/or DNA, RNA, and proteins from the same sample using commercially available column-based nucleic acid extraction kits. We further compared
the protein profile obtained with some of the methods dedicated to extracting proteins using 2-D electrophoresis, and we show that buffer choice is critical in the efficient extraction of proteins from these kits to allow proteomic studies. MATERIALS AND METHODS Tissue Preparation All experimental procedures involving human placentas were approved by the University of Newcastle Human Ethics Committee and the Hunter New England Health Human Ethics Committee. Normal term placentas were collected at John Hunter Hospital, and placental tissues were snap-frozen in liquid nitrogen prior to use. Before protein or nucleic acid extraction, samples were pulverized in liquid nitrogen. Spin Column Extractions Thirty or ten milligrams pulverized placental tissue were homogenized in
of Newcastle, New Lambton Heights, NSW, Australia and 2University of Santiago of Chile, Santiago, Chile www.biotechniques.com ı BioTechniques ı 799
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Figure 1. Representative silver stain and densitometry analysis of placental proteins extracted using conventional methods and the RNeasy kit. (A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) silver stained placental proteins: lanes 1–3, proteins conventionally extracted using radioimmunoprecipitation assay (RIPA) buffer; lanes 4–6, proteins conventionally extracted using two-dimensional (2-D) buffer; lanes 7–9, proteins extracted using RNeasy column and resuspended in RIPA buffer; lanes 10–12, proteins extracted using RNeasy column and resuspended in 2-D buffer. Molecular weight standard (Mark12; Invitrogen). (B) Representative silver stain of pooled placental proteins initially resuspended in RIPA buffer and the remaining pellet resuspended in 2-D buffer and combined. (C) Densitometrical analysis of pooled placental proteins extracted using conventional methods and the RNeasy kit. MultiGauge (Fujifilm) software was used to analyze the density of each band, and a densitometrical plot was obtained. Blue, proteins conventionally extracted using RIPA buffer; green, proteins conventionally extracted using 2-D buffer; aqua, proteins extracted using the RNeasy column and resuspended in RIPA buffer; pink, remaining pellet from proteins extracted with RNeasy using RIPA and resuspended in 2-D buffer; salmon, combined proteins extracted with RNeasy using RIPA and 2-D buffer.
buffer RLT (RNeasy mini kit; Qiagen, Doncaster, VIC, Australia) or buffer QRL1 (DNA/RNA kit; Qiagen), respectively, using the Ultra-Turrax T25 homogenizer (Janke and Kunkel IKA-Labortechnik, Staufen, Germany). Both buffers were supplemented with β-mercaptoethanol as detailed in the manufacturer’s instructions. Purification of Total RNA and Protein Using the RNeasy Mini Kit RNA was extracted using the RNeasy kit following the manufacturer’s instructions (www1.qiagen. com/literature/handbooks). The flowthrough from each of the steps was pooled, and from this, proteins were left to precipitate overnight at -20°C. Purification of DNA, RNA, and Protein Using the DNA/RNA Kit DNA and RNA were extracted using the DNA/RNA kit following the manufacturer’s instructions (www. 800 ı BioTechniques ı www.biotechniques.com
qiagen.com/literature/handbooks). The flow-through from each of the steps was pooled. The pellet from the QRV1 centrifugation step was resuspended in buffer QRL1 and added to the pooled flow-through. An equal volume of 100% ethanol was added, and the proteins were precipitated overnight at -20°C. Following extractions, the absorbance at 260 and 280 nm was measured using a NanoDrop ND-1000 spectrophotometer (Biolab, Mulgrave, VIC, Australia), and the ratios were calculated. Nucleic acid quality was further examined by agarose gel electrophoresis. Protein Extraction Conventional. Tissues were homogenized on ice using an Ultra-Turrax homogenizer in either radioimmunoprecipitation assay (RIPA) protein extraction buffer [50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxy-
cholate, 1% Triton X-100, 2 mM EDTA, 1 mM dithiothreitol (DTT), pH 7.4] supplemented with Complete Protease Inhibitor Cocktail tablets (Roche Diagnostics Australia Pty Ltd., Castle Hill, NSW Australia) or 2-D buffer (30 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS). One milliliter buffer was added per 100 mg tissue. The homogenate was then centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was collected. Column-based proteins. Precipitated flow-through from the column was spun at 10,000 × g to pellet the proteins. Protein pellets were then washed three times in acetone by incubating at -20°C for 30 min and then centrifuging the samples at 10,000 × g for 30 min. Washed protein pellets were resuspended in the appropriate buffer (RIPA or 2-D). To completely resuspend the protein pellets, samples were stored overnight at 4°C. Protein Determination Bicinchoninic acid kit. Proteins extracted using the RIPA buffer were quantified using the bicinchoninic acid (BCA) Protein Assay kit (Pierce, Rockford, IL, USA) following the manufacturer’s instructions (www. piercenet.com/files/1296dh4.pdf). 2-D Quant kit. Proteins extracted using the 2-D buffer were quantified using the 2-D Quant kit (GE Healthcare, Piscataway, NJ, USA) following the manufacturer’s instructions (www. amershambiosciences.com). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Placental proteins were resolved using NuPAGE Novex 10% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) with MOPS running buffer at 200 V for 50 min. Gels for silver staining were then fixed in silver stain fixative (40% methanol, 50% deionized water, and 10% glacial acetic acid) overnight. Gels for Western blot analysis were transferred to nitrocellulose membranes for later use. Proteins resolved by gel electrophoresis were silver stained using SilverQuest (Invitrogen), following Vol. 43 ı No. 6 ı 2007
Research Reports pH 3–10 immobilized pH gradient strips, and proteins were separated in a second dimension using 4%–12% Bis-Tris gel (Invitrogen). Following separation, proteins were visualized by Sypro Ruby Protein stain (Invitrogen) following the manufacturer’s protocol (probes.invitrogen.com/media/pis/ mp12000.pdf) and photographed using the LAS-3000.
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Figure 2. Representative silver stain and densitometry analysis of placental proteins conventionally extracted using two-dimensional (2-D) buffer or RNeasy column and DNA/RNA column and resuspended in 2-D buffer. (A) Representative silver stain of pooled placental proteins conventionally extracted using 2-D buffer and column-based methods. Molecular weight standard (Mark12; Invitrogen). Lane 1, pooled proteins conventionally extracted using 2-D buffer; lane 2, pooled proteins extracted using the DNA/RNA column and resuspended in 2-D buffer; lane 3, pooled proteins extracted using the RNeasy column and resuspended in 2-D buffer. (B) Densitometric analysis of pooled placental proteins using MultiGauge (Fujifilm) densitometrical analysis software. Blue, pooled proteins conventionally extracted with 2-D buffer; green, pooled proteins extracted with DNA/RNA columns and resuspended in 2-D buffer; aqua, pooled proteins extracted with the RNeasy columns and resuspended in 2-D buffer.
the protocol as detailed by the manufacturer (www.invitrogen. com/content/sfs/manuals/silverquest_man.pdf). To determine differences between extraction techniques, a densitometric scan was undertaken using MultiGauge software (version 2.3; Fujifilm, Tokyo, Japan). Western Blot Analysis Proteins resolved by gel electrophoresis were transferred to a nitrocellulose membrane at 25 mA for 60 min using NuPAGE transfer buffer (Invitrogen). Membranes were blocked with 5% skim milk in TBST (10 mM Tris-base, pH 7.4, 150 mM NaCl, 0.1% Tween-20, and deionized water). Membranes were then washed in TBST and immunoblotted with anti-α-actin A4700 (Sigma-Aldrich, Castle Hill, NSW, Australia) in 5% milk rocking Vol. 43 ı No. 6 ı 2007
overnight at 4°C. Membranes were washed in TBST and incubated with an anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) for 1 h rotating at room temperature. Following a final wash in TBST, α-actin was detected on the membranes using the enhanced chemiluminescence (ECL) system and photographed using the LAS-3000 (Fujifilm). 2-D Gel Electrophoresis 2-D gel electrophoresis was undertaken using the ZOOM IPGRunner System (Invitrogen) following the manufacturer’s instructions (www. invitrogen.com/content/sfs/manuals/ zoomipgrunner_man.pdf). Briefly, proteins were electrofocused in the first dimension by isoelectric focusing using
Nucleic acids extracted from the RNeasy and DNA/RNA kits were quantified using a NanoDrop spectrophotometer, and the 260:280 ratios were calculated. Nucleic acid integrity was checked using agarose gel electrophoresis. RNA ratios were shown to be between 1.8 and 2.1, while DNA ratios were between 1.7 and 2. Both DNA and RNA extracted using these techniques were of high quality, as expected from kits designed to extract nucleic acids (data not shown). One-dimensional Protein Analysis Our initial studies were designed to confirm Morse’s observations (5) by analyzing the profile of the proteins extracted with the RNeasy columns using silver staining, followed by quantitative analysis using densitometry. Our initial observation was that we were unable to completely resuspend the proteins extracted using the RNeasy column in RIPA buffer, even with heat treatment and/or overnight resuspension at 4°C. This was not the case for proteins resuspended in 2-D buffer. Protein determination of these samples showed that the RIPA buffer was inefficient in extracting proteins compared with 2-D buffer (data not shown). The profile of the proteins was then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining and densitometry. As observed in Figure 1, conventional extraction of placental proteins using RIPA or 2-D buffers yielded a wide www.biotechniques.com ı BioTechniques ı 801
Research Reports with common downstream analytical applications (Figure 3). 2-D Analysis of Proteins Extracted Using Conventional and Columnbased Methods Figure 3. Western blot analysis of placental proteins extracted using the conventional method and the column-based method. Proteins were run on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a nitrocellulose membrane. Proteins were checked for their compatibility with Western blot analysis using an anti-α-actin antibody. Lane 1, proteins conventionally extracted using two-dimensional (2-D) buffer; lanes 2 and 3, proteins extracted using the DNA/RNA columns and resuspended in 2-D buffer; lanes 4 and 5, proteins extracted using the RNeasy column and resuspended in 2-D buffer.
spectrum of proteins displaying a similar profile with some small differences, notably in the high molecular weight range (Figure 1A, lanes 1–3 and 4–6). Proteins that were extracted using the RNeasy column and resuspended in either of the two buffers also showed a wide spectrum of proteins, however, the proteins resuspended with the RIPA buffer (Figure 1A, lanes 7–9) clearly showed missing bands at certain molecular weights when compared to the conventional extraction method using RIPA (Figure 1A, lanes 1–3) or 2-D buffer (Figure 1A, lanes 4–6). Proteins from the RNeasy column resuspended in 2-D buffer (Figure 1A, lanes 10–12) displayed a highly comparable protein profile to the conventionally extracted proteins using 2-D buffer. These observations were further confirmed using densitometrical analysis (Figure 1C). As detailed above, proteins extracted using the RNeasy column were unable to be completely resuspended in the RIPA buffer. However, we showed that if the remaining pellet after resuspension in RIPA buffer was further resuspended in 2-D buffer and these samples were pooled together, a representative profile of the entire protein population could be observed. This profile was better than that observed in proteins extracted using a single buffer and contained proteins highly abundant in both RIPA and 2-D buffers (Figure 1B). Comparative Analysis of Protein Profiles Proteins were obtained using the RNeasy column, DNA/RNA 802 ı BioTechniques ı www.biotechniques.com
column, and conventional 2-D protein extraction buffer. Our initial studies confirmed Morse’s observations (5) that the RNeasy column was capable of extracting proteins following a few simple additional steps. However, we have further shown that a conventional extraction buffer such as RIPA was not compatible with this technique. In addition to the Morse study, we wished to determine whether other columnbased kits were capable of combined nucleic acids and protein extractions. Using the 2-D buffer, which we showed was highly compatible with columnbased extractions, we analyzed the capacity of the DNA/RNA column to co-extract DNA, RNA, and proteins from the same sample. Placental proteins were extracted using the conventional method with 2-D buffer (Figure 2A, lane1), the RNeasy column method (Figure 2A, lane 3), and the DNA/RNA column method (Figure 2A, lane 2). Using silver stain and densitometry, we compared the profile of the proteins at the one-dimensional (1-D) level (Figure 2, A and B). A wide range of proteins were observed, and the protein profiles were comparable between all extraction techniques when analyzed by silver staining (Figure 2A) and densitometrical analysis (Figure 2B). Western Blot Analysis To confirm the compatibility of our extraction methods with Western blot analysis, a commercial actin antibody was tested. A positive reaction was observed at 42 kDA, corresponding to the expected size for actin, showing that our extraction methods are compatible
Following our analysis of proteins simultaneously extracted with nucleic acids at the 1-D level, we decided to examine the profile of proteins at the 2-D level. The overall analysis of these gels showed a highly comparable pattern of proteins with a few exceptional differences. The most notable differences between the extraction methods were the relative abundance of some proteins preferentially extracted with one method in comparison to another. The column-based extraction methods in some cases enriched the protein profile compared to conventional extraction methods (Figure 4, red box). However, in some cases, the conventional extraction method appeared to enrich the proteins present in certain areas of the gel, particularly at low molecular weights (Figure 4). However, when the images of each 2D gel protein extraction method were matched by overlapping, the overall picture of the proteins showed a highly similar pattern. DISCUSSION Current methodologies limit the ability of the researcher to analyze nucleic acids and proteins from the same biological sample. To our knowledge, there are only a few simultaneous DNA/RNA and protein extraction techniques from the same sample (6–9), and most of these are based on variations of the Chomczynski method (7). Hummon et al. (6) recently published a methodology for the extraction of nucleic acids and protein using the TRIzol reagent based upon the above mentioned method. Using this technique, Hummon and coworkers (6) claimed that 98% of the total protein was recovered, and the protein was not degraded following long-term storage. However, Hummon’s modified technique is time consuming and requires lengthy dialysis to completely solubilize the protein. Although these Vol. 43 ı No. 6 ı 2007
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Figure 4. Two-dimensional (2-D) gels detailing proteins extracted using conventional 2-D extraction buffer, RNeasy column extraction method, and DNA/RNA column extraction method. All proteins were resuspended in 2-D buffer. Sypro Ruby was used to detect the protein spots. (A) Proteins conventionally extracted using 2-D buffer. (B) Proteins extracted using DNA/RNA columns and resuspended in 2-D buffer. (C) Proteins extracted using RNeasy columns and resuspended in 2-D buffer. Whole gels were then separated into regions for better analysis (panels D to F). Proteins were extracted using the conventional (top), DNA/RNA (middle), and RNeasy (bottom) methods (panels D to F). Boxes and circles are used to define regions where differences or similarities can be observed: (D) 66–200 kDa, (E) 31–61 kDa, (F) 6–31 kDa.
techniques allow for the simultaneous extraction of nucleic acids and proteins, new column-based extraction methods are less time consuming and require the use of less hazardous chemicals. Vol. 43 ı No. 6 ı 2007
Recently, Morse et al. (5) demonstrated that using a column designed to extract RNA (the RNeasy column), it is also possible to extract proteins, following some simple additional steps. However, Morse et al. (5) did not use
a conventional in-gel staining method to show the profile of the proteins obtained, and hence we replicated their studies. Our initial observations following resuspension of the proteins were that the RIPA buffer was unable to completely resuspend the pellet. This may be due to the fact that the wash steps were unable to completely remove the guanidine salts present from the extraction, which are incompatible with SDS-based buffers as previously reported (10). Subsequent analysis of the proteins extracted from these methods showed that RNeasy proteins resuspended in RIPA buffer had a relatively poor profile compared to those proteins extracted using the conventional method with RIPA, 2-D, and also RNeasy proteins resuspended in the 2-D buffer (Figure 1). Because of the inherent difficulties of solubilization, we suggest that prior to using this technique as a routine protocol for simultaneous extraction of nucleic acids and proteins, that the researcher compares the conventional protein extraction technique to the RNeasy extraction method using a number of different protein extraction buffers. We highly recommend that the researcher optimizes extraction protocols by varying the detergent composition of the solubilization buffer with a variety of non-ionic and zwitterionic detergents and detergentlike phospholipids when a membrane proteome analysis is involved (11). For example, Nolan and Teller (9) studied the capacity of different solvents to extract proteins from human brain. These results showed that the most effective solvent for the extraction of brain proteins was 2% diethylamine, while 2% CHAPS was found to be the least effective. We found that CHAPS was quite effective in extracting proteins from the column-based methods in human placenta. However, other buffers may provide a more optimal result depending on the tissue and proteins of interest. Following our replication of the Morse study, we wished to determine whether the DNA/RNA kit was capable of a simultaneous extraction of nucleic acids and proteins. Using this kit, all flow-through steps were pooled along www.biotechniques.com ı BioTechniques ı 803
Research Reports with the initial pellet from buffer QRV1 centrifugation step. Following an overnight incubation, the precipitated protein was centrifuged, and the same protocol as the RNeasy columns was followed. Using proteins extracted with the DNA/RNA column and resuspended in 2-D buffer, we were able to show a highly comparable protein profile to both the conventional and RNeasy extraction methods using the same buffer at the 1-D and 2-D level (Figures 2 and 4). Although we observed some differences at the 2-D level, the most important point was that all spots were present but at different intensities. We also showed that proteins extracted using these techniques were capable of being analyzed by Western blot analysis (Figure 3). In the case of the RNeasy column, no additional reagents are required to be added for precipitation. However, the DNA/RNA column flow-through requires the addition of an equal volume of ethanol prior to precipitation due to the presence of low levels of ethanol in the extraction buffers. The subsequent protein pellet was washed three times in acetone following the Morse method before air-drying the pellet and resuspending in our buffers. We strongly recommend not using trichloroacetic acid (TCA) to precipitate the proteins, due to the formation of highly reactive compounds when combining guanidine salts and acidic solutions (see www. qiagen.com/literature/protocols). There are other techniques that utilize column-based technology to simultaneously extract nucleic acids and proteins, however these techniques generally have their drawbacks. Commercially, kits such as TRI Reagent (Sigma-Aldrich) and PARIS kit (Ambion, Austin, TX, USA) allow for the dual isolation of total RNA and protein, however these methodologies are not as simple as those described by Morse et al. (5), which allows for small quantities of sample to be analyzed at the level of RNA and protein. One added bonus of using simultaneous extraction techniques is that contaminating DNA and RNA are removed from the proteins. This is particularly relevant in 2-D analysis in which contaminating nucleic acids appear as streaks on the gels. The 804 ı BioTechniques ı www.biotechniques.com
removal of these nucleic acids increases the clarity of the 2-D gels and improves protein yield and spot resolution (12). The next step of this study will be the validation of these techniques in proteomic analysis using matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-mass spectrometry (LC-MS). This will allow us to further compare the protein profile obtained between these on column protein/ nucleic acid extractions to proteomic data generated by conventional protein extraction methods. Overall, the modification of methods presented here provides a simple, safe, and effective way to simultaneously extract nucleic acids and proteins from the same sample. COMPETING INTERESTS STATEMENT The authors declare no competing interests. REFERENCES 1. Cavalcoli, J.D. 2001. Genomic and proteomic databases: large scale analysis and integration of data. Trends Cardiovasc. Med. 11:76-81. 2. Reif, D.M., B.C. White, and J.H. Moore. 2004. Integrated analysis of genetic, genomic and proteomic data. Expert Rev. Proteomics 1:67-75. 3. Cox, B., T. Kislinger, and A. Emili. 2005. Integrating gene and protein expression data: pattern analysis and profile mining. Methods 35:303-314. 4. Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159. 5. Morse, S.M.J., G. Shaw, and S.F. Larner. 2006. Concurrent mRNA and protein extraction from the same experimental sample using a commercially available column-based RNA preparation kit. BioTechniques 40:54-58. 6. Hummon, A.B., S.R. Lim, M.J. Difilippantonio, and T. Reid. 2007. Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage. BioTechniques 42:467-472. 7. Chomczynski, P. 1993. A reagent for the single-step simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples. BioTechniques 15:532-537. 8. Riol, H., B. Jeune, A. Moskovic, L. Bathum, and E. Wang. 1999. Optimized
lymphocyte protein extraction performed simultaneously with DNA and RNA isolation: application to the study of factors affecting DNA, RNA and protein recovery from lymphocytes of the oldest individuals. Anal. Biochem. 275:192-201. 9. Nolan, R.L. and J.K. Teller. 2006. Diethylamine extraction of proteins and peptides isolated with a mono-phasic solution of phenol and guanidine isothiocyanate. J. Biochem. Biophys. Methods 68:127-131. 10. Banerjee, S., A. Smallwood, A.E. Chambers, and K. Nicholaides. 2003. Quantitative recovery of immunoreactive proteins from clinical samples following RNA and DNA isolation. BioTechniques 35:450-456. 11. Churchward, M.A., R. Hussain Butt, J.C. Lang, K.K. Hsu, and J.R. Coorssen. 2005. Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis. Proteome Sci. 3:5. 12. Kirkland, P.A., J. Busby, S. Stevens, Jr., and J.A. Maupin-Furlow. 2006. Trizolbased method for sample preparation and isoelectric focusing of halophilic proteins. Anal. Biochem. 351:254-259.
Received 8 July 2007; accepted 24 August 2007. Address correspondence to Jorge M. Tolosa, Mothers and Babies Research Centre, Level 3, Endocrine Unit, John Hunter Hospital, New Lambton Heights, 2305 NSW, Australia. e-mail: jorge.tolosa
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