One-dimensional Polyacrylamide Gel Electrophoresis Research Articles

Development of CuO Nanoparticles from the Mucus of Garden Snail Cornu aspersum as New Antimicrobial Agents.

Several biologically active compounds involved in the green synthesis of silver and gold nanoparticles have been isolated from snail mucus and characterized. This paper presents a successful method for the application of snail mucus from Cornu aspersum as a bioreducing agent of copper sulfate and as a biostabilizer of the copper oxide nanoparticles (CuONPs-Muc) obtained. The synthesis at room temperature and neutral pH yielded nanoparticles with a spherical shape and an average diameter of 150 nm. The structure and properties of CuONPs-Muc were characterized using various methods and techniques, such as ultraviolet-visible spectroscopy (UV-vis), high-performance liquid chromatography (HPLC), one-dimensional polyacrylamide gel electrophoresis (1D-PAGE), up-conversion infrared spectroscopy Fourier transform (FTIR), scanning electron microscopy combined with energy dispersive spectroscopy (SEM/EDS), Raman spectroscopy and imaging, thermogravimetric analysis (TG-DSC), etc. Mucus proteins with molecular weights of 30.691 kDa and 26.549 kDa were identified, which are involved in the biogenic production of CuONPs-Muc. The macromolecular shell of proteins formed around the copper ions contributes to a higher efficiency of the synthesized CuONPs-Muc in inhibiting the bacterial growth of several Gram-positive (Bacillus subtilis NBIMCC2353, Bacillus spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, Listeria innocua NBIMCC8755) and Gram-negative (Escherichia coli ATCC8739, Salmonella enteitidis NBIMCC8691, Salmonella typhimurium ATCC 14028, Stenotrophomonas maltophilia ATCC 17666) bacteria compared to baseline mucus. The bioorganic synthesis of snail mucus presented here provides CuONPs-Muc with a highly pronounced antimicrobial effect. These results will expand knowledge in the field of natural nanomaterials and their role in emerging dosage forms.

Salt and drought stress exhibits oxidative stress and modulated protein patterns in roots and leaves of date palm (Phoenix dactylifera L.)

The formation of new proteins under the influence of harsh environmental conditions is a plant adaptation reaction. Two-year-old date palm tissue culture-derived plants from ‘Barhee’ grown in the field were subjected to salt stress (70 g l-1 NaCl) and dehydration-induced by applying 70 g l-1 polyethylene glycol or without irrigation and withholding irrigation (0 g l-1) for one month. The soluble carbohydrate content increased in response to salinity and polyethylene glycol treatment in leaves compared to the control and drought treatment without irrigation. Proline increased in all treatments. Malondialdehyde and hydrogen peroxide increased under salinity. Salinity treatment increased the activity of ascorbate peroxidase and catalase enzyme. Salinity and polyethylene glycol treatments increased abscisic acid, whereas the indoleacetic acid level decreased. The protein pattern of roots and leaves in one-dimensional polyacrylamide gel electrophoresis showed that the stress conditions led to new protein bands' appearance and other proteins' disappearance. A comparison of protein patterns between the control and stress treatments revealed that the relative intensity of proteins in roots and leaves were more associated with salinity treatment than the drought. The results may be clearing important the molecular mechanism of tolerance under the influence of extreme environmental stress.

Comparative two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of human milk to identify dysregulated proteins in breast cancer.

Breast cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC-associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot proteomics study, using one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her milk. Statistically different gel spots were picked for protein digestion followed by nanoliquid chromatography tandem MS (nanoLC-MS/MS) analysis. The upregulated proteins in BC versus control are alpha-amylase, gelsolin isoform a precursor, alpha-2-glycoprotein 1 zinc isoform CRA_b partial, apoptosis-inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease. MS data is available via ProteomeXchange with identifier PXD009860.

IDENTIFICATION OF IMMUNOGENIC PROTEINS OF STRAINS OF BACILLUS ANTHRACIS IN MALDI TOF MS

Aim. Identification of obtained in host-simulated conditions immunogenic proteins of isogenic variants of Bacillus anthracis 575/122. Materials and methods. We used culture filtrate of isogenic variants of B. anthracis 575/122: R02 (pXOL pXO2+); R01 (pXO1+ pXO2‘); R00 (pXOL pX02~), obtained in host-simulated conditions. In the one-dimensional polyacrylamide gel electrophoresis and immunoblotting with hyperimmune serums immunodominant proteins, that have been identified in MALDI TOF MS. Results. Immunoblotting revealed proteins with molecular masses in range 97 - 14.1 kDa. 90 kDa protein from strain B. anthracis 575/122 R01 in MALDI TOF MS was identified as protective antigen with 85.810 kDa. Protein with molecular mass 60 kDa was identified as GMP synthase with molecular mass 57.239 kDa. In the culture filtrates of three strains two common antigen were identified: protein with molecular mass 97 kDa, identified as B. anthracis EA 1 with molecular mass 91.361 kDa protein and 45 kDa protein as enolase B. anthracis with molecular mass 46.418 kDa. Conclusion. Thus, the conditions that simulate the host can promote the production of immunodominant proteins of B. anthracis. The data about molecular-weight characteristics of protective antigen and EA 1 protein as well as some of proteases of B. anthracis are confirmed by the MALDI TOF MS. The results can be used for isolation of these proteins to improve the diagnostic and vaccine preparations.

Investigation of Differentially Expressed Proteins of Candida tropicalis Biofilm in Response to Citral

Candida tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. C. tropicalis biofilms can cause persistent infections which are difficult to treat due to acquired resistance. Citral has been used as antifungal agents against Candida species and biofilms. In the present study, we used one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF-MS) to identify the changes in the protein expression of C. tropicalis in response to the sub-lethal concentration of citral. A total of six differential proteins involved inoxidative stress (Tsa1p, Psa2p), amino acid biosynthesis (Met6p, Gln1p), heme biosynthesis (Hem13p) and glucose metabolism (Eno1p) pathways were detected. Our results revealed citral-induced proteins of C. tropicalis biofilm. This study will further help in the interpretation of mode of action of citral and development of novel antifungal agents against these potential protein targets.

Electrophoretic Detection and Confocal Microscopic Imaging of Tyrosine Nitrated Proteins in Plant Tissue.

Tyrosine nitrated proteins can be detected in plant cells electrophoretically and their distribution can be monitored by confocal laser scanning microscopy (CLSM) imaging. One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by Western blotting using polyclonal antibody against 3-nitrotyrosine residues enables detection of tyrosine nitrated proteins in plant cells. Here we describe detection of tyrosine nitrated proteins in the homogenates derived from sunflower (Helianthus annuus L.) seedling cotyledons. Total soluble proteins obtained from tissue homogenates are resolved using vertical gel electrophoresis followed by their electrophoretic transfer on to a microporous membrane support for immunodetection. Spatial distribution of tyrosine nitrated proteins can be visualized using an antibody against 3-nitrotyrosine residues. Immunofluorescent localization is performed by cutting 7μm thick wax sections of tissue followed by incubation in primary anti-nitrotyrosine antibody (dilution 1:200) and secondary Cy-3 labeled anti-rabbit IgG antibody (dilution 1:1500). Confocal laser scanning microscopy analysis is undertaken using argon lasers (ex: 530-550 nm and em: 570nm) at pinhole 1. Modulation in the abundance and spatial localization of tyrosine nitrated proteins in plant tissues can be monitored using these techniques.

Protocol for the Analysis of Laser Capture Microdissected Fresh-Frozen Tissue Homogenates by Silver-Stained 1D SDS-PAGE.

The heterogeneity present in solid tumors adds significant difficulty to scientific analysis and improved understanding. Fundamentally, solid tumor formation consists of cancer cells proper along with stromal elements. The burgeoning malignant process is dependent upon modified stromal elements. Collectively, the stroma forms an essential microenvironment, which is indispensable for the survival and growth of the malignant neoplasm. This cellular heterogeneity makes molecular profiling of solid tumors via mass spectrometry (MS)-based proteomics a daunting task. Laser capture microdissection (LCM) is commonly used to obtain distinct histological cell types (e.g., tumor parenchymal cells, stromal cells) from tumor tissue and attempt to address the tumor heterogeneity interference with downstream liquid chromatography (LC) MS analysis. To provide optimal LC-MS analysis of micro-scale and/or nano-scale tissue sections, we modified and optimized a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) protocol for the LC-MS analysis of LCM-procured fresh-frozen tissue specimens. Presented is a detailed in-gel digestion protocol adjusted specifically to maximize the proteome coverage of amount-limited LCM samples, and facilitate in-depth molecular profiling. Following LCM, targeted tissue sections are further fractionated using silver-stained 1D-SDS-PAGE to resolve and visualize tissue proteins prior to in-gel digestion and subsequent LC-MS analysis.

Blue Native PAGE Analysis of Bacterial Secretion Complexes.

Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.

Protein profiling analysis of Gossypium hirsutum (Malvales: Malvaceae) leaves infested by cotton whitefly Bemisia tabaci (Homoptera: Aleyrodidae)

The whitefly Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) is an important pest of cotton in many countries including Pakistan. Even though it has gained resistance for various registered insecticides such as the neonicotinoid insecticide thiamethoxam, the cellular changes occurring in plants during whitefly infestation and resistance are poorly understood. In particular, the proteomic analysis of interaction between cotton plant and devastating whiteflies is poorly understood. To reveal the physiological response as well as the molecular interaction during the incursion of B. tabaci, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D SDS-PAGE) following liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was conducted, combined with in silico protein characterizations and protein–protein interaction following validation of LC–MS/MS results by quantitative real-time PCR. The shotgun proteomic analysis of cotton leaves infested with whitefly resulted in the identification of 38 proteins, and 24 under control (non-infested) conditions. Of them, 11 proteins were identified as shared proteins, 13 were differentially induced in non-infested conditions, and 27 were induced only under whitefly-infested conditions. The major proteins like MAP kinases, COBRA-like protein family, and NBS disease resistance protein were expressed under infested conditions; while the heat shock protein 90 and cyclic nucleotide-gated ion channel 5-like major proteins were expressed under whitefly non-infested conditions. The current manuscript presents new insights into the mechanism of whitefly infestation and our understanding of the roles of COB and COBLs in host-pest interaction. To the best of our knowledge, this is the first ever report to reveal the role of several proteins, including COB and COBLs in host-pest leaf interaction.

Changes in the OXPHOS system in leaf and root mitochondria of Arabidopsis thaliana subjected to long-term sulphur deficiency

Long-term sulphur (S) deficiency in Arabidopsis thaliana affects the functioning of the mitochondrial oxidative phosphorylation system (OXPHOS) via alteration of the multisubunit NADH-ubiquinone oxidoreductase (Complex I; EC 1.6.5.3), which contains several iron–sulphur clusters. Densitometric analysis of bands of respiratory chain complexes after one-dimensional blue-native polyacrylamide gel electrophoresis (BN-PAGE) showed that levels and in-gel capacities of Complex I in leaf and root mitochondria were lower than those of the control. Two-dimensional BN/SDS-PAGE showed lower abundance of all Complex I subunits, but the qualitative structural composition (subunit expression and mobility) did not change. In mitochondria of S-deficient A. thaliana, impairment of Complex I could be compensated to some extent by additional type II NADH dehydrogenases that do not contain iron–sulphur clusters. The level and capacity of external NADH dehydrogenases in leaf and root mitochondria was higher under S deficiency, but that of internal NADH dehydrogenases did not differ from the control. The amount of COXII (mitochondrial-encoded subunit of cytochrome c oxidase in Complex IV; EC 1.9.3.1) and the capacity of Complex IV were lower under S deficiency, but levels of alternative oxidase, a bypass to Complex IV, did not change. We discuss S deficiency in A. thaliana in relation to the assembly and stability of Complex I and to a bypass of Complex I by external type II NADH dehydrogenases.

Rapid Detection of Proteins on One-Dimensional Polyacrylamide Gel Electrophoresis Using an Inexpensive Fluorescent Optical Brightener

A commercial fabric fluorescent optical brightener Ranipal® (F-OB), has been successfully employed to stain proteins on native and SDS-1D-PAGE. The F-OB was purified by using a biphasic solvent system of dichloromethane and water. The Rf value (0.63) of purified and crude F-OB were comparable on TLC. The mass spectrometry of purified F-OB indicated base peak at 414 (m/z). Absorption and emission maxima of F-OB were found to be 350 nm and 430 nm, respectively. The F-OB could stain the proteins both pre- and post-electrophoretic run, on native gel. Postelectrophoretic staining was rapid and required 20 min to visualize the stained proteins. On the other hand, in SDS gels, an additional 20 min was required for the extraction of SDS before staining the proteins with F-OB. SDS was found to interfere with binding of F-OB to proteins. Varying concentrations of molecular weight markers were loaded and their fluorescent intensity was plotted against the concentration of the proteins. The r2 values ranged from 0.965 to 0.997 indicating excellent linearity. The detection for carbonic anhydrase was in the range of 8.0-800 ng. Unlike most of the dyes used for protein staining, staining with F-OB could be carried out in tank buffer (2 mg/100 mL) and was also reversible. The F-OB, perhaps would be the most cost-effective fluorescent dye to stain the proteins (US $ 0.04/25.0 g). The F-OB was found to be simple, safe, sensitive, less time consuming and economical fluorescent dye as an alternative, for staining proteins on polyacrylamide gels.

A visual sensor array for pattern recognition analysis of proteins using novel blue-emitting fluorescent gold nanoclusters.

This paper describes a visual sensor array for pattern recognition analysis of proteins based on two different optical signal changes: colorimetric and fluorometric, by using two types of novel blue-emitting collagen protected gold nanoclusters and macerozyme R-10 protected gold nanoclusters with lower synthetic demands. Eight proteins have been well-discriminated by this visual sensor array, and protein mixtures after one-dimensional polyacrylamide gel electrophoresis also could be well-discriminated. The possible mechanism of this sensor array was illustrated and validated by fluorescence spectra, X-ray photoelectron spectroscopy (XPS), fluorescence lifetime, isothermal titration calorimetry (ITC), and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) experiments. It was attributed to that the adsorption of proteins onto the surface of gold nanoclusters (Au NCs), forming the protein-Au NCs complex. Furthermore, serums from normal and hepatoma patients were also effectively discriminated by this visual sensor array, showing feasible potential for diagnostic applications.

Transgelin: An androgen-dependent protein identified in the seminal vesicles of three Saharan rodents

During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP21 (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP21 by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP21 (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP21 (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP21, MSSVP21, and MCSVP21 seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms.

Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.

Through the eye of an electrospray needle: mass spectrometric identification of the major peptides and proteins in the milk of the one‐humped camel (Camelus dromedarius)

The milk of the one-humped camel (Camelus dromedarius) reportedly offers medicinal benefits, perhaps because of its unique bioactive components. Milk proteins were determined by (1) two-dimensional gel electrophoresis and peptide mass mapping and (2) liquid chromatography-tandem mass spectrometry (LC-MS/MS) following one-dimensional polyacrylamide gel electrophoresis. Over 200 proteins were identified: some known camel proteins including heavy-chain immunoglobulins and others exhibiting regions of exact homology with proteins from other species. Indigenous peptides were also identified following isolation and concentration by two strategies: (1) gel-eluted liquid fraction entrapment electrophoresis and (2) small-scale electrophoretic separation. Extracts were analyzed by LC-MS/MS and peptides identified by matching strategies, by de novo sequencing and by applying a sequence tag tool requiring similarity to the proposed sequence, but not an exact match. A plethora of protein cleavage products including some novel peptides were characterized. These studies demonstrate that camel milk is a rich source of peptides, some of which may serve as nutraceuticals.

Determination of Metal Associated with Proteins of Wheat Seed Samples After Sequential Extraction Procedure

This work was initially conducted to study the distribution of Cu, Fe, Mn and Zn in the protein fractions of wheat sample subjected to sequential extraction procedure. The highest metal concentrations were determined in the fraction extracted with Tris-HCl buffer containing 1% sodium dodecyl sulfate (SDS). The fractions of wheat sample obtained after the extraction procedure were analyzed by one-dimensional polyacrylamide gel electrophoresis (1D SDS-PAGE) and protein bands were treated to quantify Cu, Fe, Mn, and Zn by graphite furnace atomic absorption spectrometry (GF AAS). Metals were associated to protein bands of specific molecular weight, determined using a standard protein. The protein fractions were also analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after a typical protein digestion protocol, identifying lipid transfer protein and grain softness, the latter being possibly associated to Mn.

Cod (Gadus morhua) Muscle Proteome Cataloging Using 1D-PAGE Protein Separation, Nano-Liquid Chromatography Peptide Fractionation, and Linear Trap Quadrupole (LTQ) Mass Spectrometry

Because Atlantic cod (Gadus morhua) has high economic value and its protein-rich muscle tissue is a food source, an increased understanding of the effects and consequences of environmental, nutritional, biological, and industrial factors on meat quality is necessary. To gain insight into cod muscle tissue protein composition, a large-scale proteomics approach has been used. One-dimensional polyacrylamide gel electrophoresis, nanoflow liquid chromatography peptide separation, and linear trap quadrupole mass spectrometry were used to identify 4804 peptides, which retrieved 9113 cod expressed sequence tags (ESTs), which in turn were mapped to 446 unique proteins. The same data set identified 3924 proteins from the zebrafish protein database, which highlights the complementary value of the two approaches. The generated data sets will act as a foundation for studies related to physiological status assessment of cod under different environmental conditions, screening for diseases, and biomarker identification for assessment of fish quality during industrial processing and preservation.

Existence of hybrid structures in cationic liposome/DNA complexes revealed by their interaction with plasma proteins

The self-assembling of cationic liposomes (CLs) and DNA can give rise to a variety of nanostructures and morphologies. Multilamellar complexes are made of DNA intercalated between opposing lipid bilayers, while clusters formed by intact vesicles may exist as ‘beads-on-a-string’ (i.e. CLs attached to a string of DNA). Precise knowledge of the structure and morphology of complexes is relevant in many biological important processes such as gene delivery. Here dynamic light scattering, zeta potential, synchrotron small angle X-ray scattering and one-dimensional polyacrylamide gel electrophoresis were performed to investigate the equilibrium structure, morphology and interactions with plasma proteins of lipoplexes made of the cationic lipid (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl])-cholesterol (DC-Chol), the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE) and DNA. Results show that DC-Chol–DOPE/DNA complexes are multilamellar systems with DNA protected by cationic lipids. On the other hand, the ‘protein corona’ associated to lipoplexes after interaction with human plasma was found to be much richer in basic immunoglobulins gamma proteins (Ig-Gs) than that of pure lipid vesicles in the absence of DNA. According to the most recent evidences reported in the literature, this finding would suggest the existence of hybrid structures made of multilamellar complexes either stuck together by DNA or coexisting with DNA-loaded intact vesicles. Because surface properties of lipoplexes may determine their interaction with cells and tissues, these results may be important for predicting biological responses in vivo.

An efficient extraction method to enhance analysis of low abundant proteins from soybean seed

Large amounts of the major storage proteins, β-conglycinin and glycinin, in soybean ( Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the low abundant proteins, different from the main storage protein fractions, in one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of isopropanol showed that greater than 30% isopropanol was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing isopropanol concentration above 40% resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography–mass spectrometry (LC–MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.

Demonstration of Basic Proteins That Bind Retinoic Acid in the Human Myeloid Leukemia Cell Line HL60

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA is a potent anticancer agent that induces differentiation of leukemia cells as well as inhibiting cell growth. The current study examined HL60 proteins using anti-RA monoclonal antibodies (ARMAs) and found that some RA-binding proteins may be histones. These proteins eluted in the void volume fractions following Mono Q anion exchange chromatography and immunostained with ARMAs. These ARMAs showed specific binding to the proteins in a saturable manner that depended on antibody concentration. Certain of these proteins co-migrated with histones on one-dimensional polyacrylamide gel electrophoresis. It was also found that histones isolated from HL60 cells treated with RA also immunostained with ARMAs. These results indicate that basic proteins, including histones, may be bound to RA covalently in HL60 cells and that RA-binding histones may play significant roles in the transcriptional regulation of genes by RA.