2-D PAGE Map Analysis: Methods and Protocols

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The present study employed an alkaline-based method for the efficient isolation of hair proteins and subsequently examined them using gel-based proteomics. The introduction of two proteomic protocols, namely the conventional and modified protocol, have resulted in the detection of more than protein spots on the two-dimensional gel electrophoresis 2DE.

When compared, the modified protocol is deemed to improve overall reproducibility, whilst offering a quick overview of the total protein distribution of hair. The development of this high-performance protocol is hoped to provide a new approach for hair analysis, which could possibly lead to the discovery of biomarkers for hair in health and diseases in the future. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Proteomics has been extensively used over the last decade due to its ability to unveil the proteome of a cell, tissue, or organism and explicitly exhibit their dynamic states [ 1 ]. However, this high-throughput technique was seldom used to describe the proteome of human hair shaft. The study of such simple and stable specimen has not been broadly discussed from the proteomics standpoint until recent years.

This was due to the difficulty in extraction of hair protein caused by its high stability and keratin content, which often remains insolubilized even in strong denaturants [ 2 ]. These keratins, together with small hydrophobic proteins, known as keratin-associated proteins KAPs , make up the primary structural of the human hair [ 3 ]. Besides, the insolubility of hair was also a result of the extensive disulfide bond cross-linking between KAPs and keratin intermediate filaments KIFs , which exist in cytoplasm of hair cortical cells [ 4 ].

The interaction between KIFs and KAPs is not only crucial for hair growth, but also to provide tensile strength and elasticity to the hair [ 5 , 6 ]. Several efforts have been made to extract hair protein, followed by identification of proteins present in human hair. To date, over proteins were identified [ 7 ]. Nonetheless, the study of human hair shaft proteins by gel-based proteomics was scarcely reported.

The study also examined the composition of the extracted protein using 2DE and resulted in at least two spots in the more acidic region together with at least three spots from the acidic to basic regions [ 8 ]. Some proteins also appeared as unfocused horizontal streaks, which may be caused by the lack of sample clean-up and protein precipitation that contributed to high salt ions in the sample [ 9 ].

Hence, it is considered obligatory to remove potential impurities and interfering substances prior to sample application in order to attain desirable 2DE gel [ 10 ]. Even so, there is possible loss of protein during precipitation [ 11 ] that could eventually result in inadequate sample at undetectable level. Moreover, it is also believed that high-abundant proteins may impede the appearance of low-abundant proteins in the sample [ 12 ].

The study of post-translational modifications and protein abundances of hair is essential for the thorough understanding of human hair. This is feasible through the use of 2DE [ 13 ]. This classical method could separate proteins based on their charge as well as molecular size in order to generate protein expression profiles [ 14 ]. A representative view of hair complexity could be obtained, which would then be useful for comparative as well as protein expression analysis.

Overcoming the challenges in hair protein extraction is therefore imperative. The current study has selected an alkaline-based method to obtain hair proteins from hair shaft samples. Previous study has demonstrated efficient extraction of hair protein by heating short strands of hair in an alkaline lysis buffer [ 15 ]. Subsequently, the quality of the extracted protein by this methodology and its compatibility with the downstream processes were assessed in this study.

Through the use of this method of extraction, adequate protein was attainable to be resolved in both first and second dimensional gel electrophoresis. Following the separation of hair protein across a two-dimensional gel using conventional proteomic techniques, the present study then applied some modifications on these sophisticated techniques to overcome some limitations that were found in the conventional protocol.

The comparison between these protocols including the difference in sample preparation, isoelectric focusing IEF as well as 2DE was made to determine the most suitable method to reveal information of such complex tissue—hair. The establishment of this high-performance human hair shaft proteomics is essential for comparative analysis in the future, where gel-to-gel reproducibility is often a point in question.

In addition to providing an insight into hair proteome profile, the adoption of these high-throughput techniques in the modified protocol could serve as a novel approach to discover potential biomarkers of hair health, ageing and various diseases in the future. Conventional proteomic techniques and protocol mentioned throughout this study were carried out in the Department of Molecular Medicine of University of Malaya, Malaysia [ 15 ].

However, the second stage of extraction was omitted in this study to reduce the total extraction duration. Prior to applying samples for gel electrophoresis, the concentration of protein in both samples were determined by Bradford colorimetric method or 2D Quant kit GE Healthcare, FairField, CT. In the conventional protocol, protein solution was mixed with rehydration solution which has similar constituent to the sample buffer prepared earlier, except a few grains of DTT was only added prior to use.

A few grains of Orange G as tracking dye was also added. In both experiments, the volumes of protein solution added were based on the result of protein quantification. Moreover, different types of Immobiline DryStrips 13 cm, pH 4—7; 7 cm, pH 3—10 were also compared in this study.

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The former was adopted in the conventional protocol. It was performed by gradually increasing the voltage across the DryStrip under the following conditions: i 0— V, 1 h; ii V, 1 h; iii V, 2 h 30 m, and iv V, 55 m.

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For the modified protocol, first-dimensional separation of protein was done using similar power supply, except Multiphor II GE Healthcare and a cooling circulator Julabo, Seelbach, Germany was linked. It was allowed to run for at least 5 hours until the current value became constant. The strip was immediately used for the next step. The modified method also utilized equilibration buffer with the same components, except the concentrations of Tris-HCl and glycerol were altered to 1.

Moreover, the DryStrip was only incubated for 15 m each at RT. The comparison between hand-cast and pre-cast gels, as well as different SDS-PAGE gel percentages and sizes were also made in this study.

Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis

Agarose sealing solution of 0. Similarly, 0. The electrophoresis was stopped when the marker reached the mark above the protrusion of the gel cassette. Two different gel-staining methods were compared in the current study: silver-staining for conventional protocol; Sypro Ruby-staining for modified protocol.

The washing step was repeated thrice for 5 min each. Next, the gel was impregnated with silver solution The gel was later incubated in developing buffer 0. When sufficient degree of spot intensities has been achieved, staining is stopped using stop solution [40 mM ethylenediaminetetraaacetic acid EDTA sodium dihydrate] for 30 min before rinsing twice with distilled water. This silver-staining method was performed on an orbital shaker BioLab, UbiTechPark, Singapore which was set at a constant speed of 50 rpm.

It is crucial to evaluate the appropriate assay of protein quantification, as well as the method for sample clean-up to suit the experimental design in this study. The conventional protocol utilized pre-cold acetone for protein precipitation and resulted in 5. Although the amount of extracted proteins in both experiments were comparable, the efficacy of each clean-up method should be conferred altogether with the resulted 2DE gel profiles, which will be further discussed later in this study.

In both experiments prior to quantification, protein pellets were reconstituted in either sample buffer or Destreak Rehydration solution. When sample buffer containing DTT was used, the protein pellets were completely dissolved. On the other hand, in the case where Destreak Rehydration solution was used, some parts of the protein pellets obtained from 2D cleaned-up sample remained jelly-like, which was resistant to solubilization. The current study has successfully obtained 2DE gel profiles of human hair through both the conventional and modified protocols.

The distribution of spots of hair proteins is displayed in Fig 1. Spot detection was also performed on these gels using Image Master 2D Platinum Software and the result is displayed in Table 1. A Conventional protocol. In biological membranes, many proteins are organized in complexes. The blue native electophoresis protocol is used to determine the size, relative abundance and subunit composition of protein complexes.

Protein complexes organize and maintain the cellular and organelle functions on all levels of complexity in time and space, including cell development and division, transcription and translation, respiration and photosynthesis, transport and metabolism. Protein complexes may exhibit partial and temporal changes, as well as exhibit different stabilities, making the identification and analysis extremely difficult. Coomassie blue does not act as a detergent, and it preserves the structure of protein complexes. Comprehensive chromatographic separations in proteomics.

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The principle of 2D Gel Electrophoresis/and the isoelectric point

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Golden, Zhiqun Zhang, William E. Hayes, Kevin K. Wang, Nancy D. Neuroproteomics in neurotrauma.


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2-D PAGE Map Analysis: Methods and Protocols 2-D PAGE Map Analysis: Methods and Protocols
2-D PAGE Map Analysis: Methods and Protocols 2-D PAGE Map Analysis: Methods and Protocols
2-D PAGE Map Analysis: Methods and Protocols 2-D PAGE Map Analysis: Methods and Protocols
2-D PAGE Map Analysis: Methods and Protocols 2-D PAGE Map Analysis: Methods and Protocols
2-D PAGE Map Analysis: Methods and Protocols 2-D PAGE Map Analysis: Methods and Protocols

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