Bovine DNA Research Articles

Investigating the Quantification Capabilities of a Nanopore-Based Sequencing Platform for Food Safety Application via External Standards of Lambda DNA and Lambda Spiked Beef.

Six hundred million cases of disease and roughly 420,000 deaths occur globally each year due to foodborne pathogens. Current methods to screen and identify pathogens in swine, poultry, and cattle products include immuno-based techniques (e.g., immunoassay integrated biosensors), molecular methods (e.g., DNA hybridization and PCR assays), and traditional culturing. These methods are often used in tandem to screen, quantify, and characterize samples, prolonging real-time comprehensive analysis. Next-generation sequencing (NGS) is a relatively new technology that combines DNA-sequencing chemistry and bioinformatics to generate and analyze large amounts of short- or long-read DNA sequences and whole genomes. The goal of this project was to evaluate the quantitative capabilities of the real-time NGS Oxford Nanopore Technologies' MinION sequencer through a shotgun-based sequencing approach. This investigation explored the correlation between known amounts of the analyte (lambda DNA as a pathogenic bacterial surrogate) with data output, in both the presence and absence of a background matrix (Bos taurus DNA). A positive linear correlation was observed between the concentration of analyte and the amount of data produced, number of bases sequenced, and number of reads generated in both the presence and absence of a background matrix. In the presence of bovine DNA, the sequenced data were successfully mapped to the NCBI lambda reference genome. Furthermore, the workflow from pre-extracted DNA to target identification took less than 3 h, demonstrating the potential of long-read sequencing in food safety as a rapid method for screening, identification, and quantification.

Under Water Superelastic Porous Nanofibrous Sponge for Efficient RNA Separation and Purification.

Developing monolithic materials for chromatography columns with a novel interconnected porous structure is vital for the enhancement of the separation efficiency of RNA purification processes. Herein, a porous nanofibrous sponge (PNFS) is constructed by freeze molding and freeze-drying a nanofiber dispersion with ethylene vinyl alcohol copolymer nanofibers as the skeleton, chitosan (CS) and polyethylenimine (PEI) as the binders, and glutaraldehyde (GA) as the crosslinking agent. The results show that when the CS content of the dispersion is 1.5 wt %, PNFS demonstrates a high static adsorption capacity of 406.5 mg/g (30.7 mg/m2) and a dynamic adsorption capacity of 382.6 mg/g (28.9 mg/m2) at a flow rate of 1 mm/min. Moreover, PNFS shows a high specific adsorption performance toward RNA in the presence of bovine serum albumin, lecithin, or DNA by adjusting the solution pH value and the method of gradient elution. Besides, PNFS presents exceptional performance in the rapid separation of RNA from HT22 cells without degradation. This result can be attributed to optimized morphology, pore structure, and comprehensive performance of PNFS, benefiting from the synergistic effect of the highly oriented porous structure and CS-PEI interaction derived from the high-density adsorption ligands on the channel walls of PNFS. This work provided an efficient strategy to handle the permeability/adsorptivity trade-off for ion-exchange chromatographic materials.

Novel Copper (II) Complexes with Fluorine-Containing Reduced Schiff Base Ligands Showing Marked Cytotoxicity in the HepG2 Cancer Cell Line.

Several novel copper (II) complexes of reduced Schiff bases containing fluoride substituents were prepared and structurally characterized by single-crystal X-ray diffraction. The complexes exhibited diverse structures, with the central atom in distorted tetrahedral geometry. The biological effects of the products were evaluated, specifically their cytotoxicity, antimicrobial, and antiurease activities, as well as affinity for albumin (BSA) and DNA (ct-DNA). The complexes showed marked cytotoxic activities in the HepG2 hepatocellular carcinoma cell line, considerably higher than the standard cisplatin. The cytotoxicity depended significantly on the substitution pattern. The best activity was observed in the complex with a trifluoromethyl group in position 4 of the benzene ring-the dichloro[(±)-trans-N,N'-bis-(4-trifluoromethylbenzyl)-cyclohexane-1,2-diamine]copper (II) complex, whose activity (IC50 28.7 μM) was higher than that of the free ligand and markedly better than the activity of the standard cisplatin (IC50 336.8 μM). The same complex also showed the highest antimicrobial effect in vitro. The affinity of the complexes towards bovine serum albumin (BSA) and calf thymus DNA (ct-DNA) was established as well, indicating only marginal differences between the complexes. In addition, all complexes were shown to be excellent inhibitors of the enzyme urease, with the IC50 values in the lower micromolar region.

2-Propyl-1H-imidazole-4,5-dicarboxylate acid supported cu/ni/fe/co complexes: synthesis, biological and catalytic evaluation

Herein, we report 2-propyl-1H-imidazole-4,5-dicarboxylate complexes of Cu/Co/Fe/Ni metal ions. The complexes have been characterized using powder X-ray diffraction, FTIR, and mass spectrometry. Thermal gravimetric analysis (TGA) established their thermal stability, enduring temperatures of up to 650 °C. The interaction of Bovine Serum Albumin (BSA) and DNA with these complexes was examined, revealing a significant binding affinity up to 70%. Fluorescence quenching experiments also showed the binding of complexes with biomolecules. These interactions were substantiated by molecular docking studies against the proteins 3v03 (BSA) and 1d28 (DNA), shedding light on the essential interactions driving their potential medicinal activity. The Cu complex displayed a notably high GSC score and a substantial area as determined through the Patch Dock server. Free radical interaction with 2,2-diphenylpicrylhydrazyl (DPPH) shows their antioxidant activity where Cu and Fe complexes expressed greater activity as compared to others. The catalytic properties of the compounds were assessed using p-nitrophenol (PNP) where the complexes exhibited reducing activity, leading to a conversion of over 90% of PNP into p-aminophenol. Analysis revealed a pseudo-second-order model closely aligned with experimental values, indicating second-order reduction kinetics.

Copper(II) and Zinc(II) Complexes with Bacterial Prodigiosin Are Targeting Site III of Bovine Serum Albumin and Acting as DNA Minor Groove Binders.

The negative environmental and social impacts of food waste accumulation can be mitigated by utilizing bio-refineries' approach where food waste is revalorized into high-value products, such as prodigiosin (PG), using microbial bioprocesses. The diverse biological activities of PG position it as a promising compound, but its high production cost and promiscuous bioactivity hinder its wide application. Metal ions can modulate the electronic properties of organic molecules, leading to novel mechanisms of action and increased target potency, while metal complex formation can improve the stability, solubility and bioavailability of the parent compound. The objectives of this study were optimizing PG production through bacterial fermentation using food waste, allowing good quantities of the pure natural product for further synthesizing and evaluating copper(II) and zinc(II) complexes with it. Their antimicrobial and anticancer activities were assessed, and their binding affinity toward biologically important molecules, bovine serum albumin (BSA) and DNA was investigated by fluorescence emission spectroscopy and molecular docking. The yield of 83.1 mg/L of pure PG was obtained when processed meat waste at 18 g/L was utilized as the sole fermentation substrate. The obtained complexes CuPG and ZnPG showed high binding affinity towards target site III of BSA, and molecular docking simulations highlighted the affinity of the compounds for DNA minor grooves.

Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

Green synthesis of iron oxide nanoparticles using Nicotiana plumbaginifolia and their biological evaluation

Herein, we report an environmentally friendly synthesis of the iron oxide nanoparticles (FeO_NPs) employing Nicotiana plumbaginifolia (NiP) leaf extract, which was then examined using UV, FTIR, TGA/ DSC, and SEM tools. Their antimicrobial activity (AMA) was assessed against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa microorganisms, where they have shown good AMA demonstrated by zone of inhibition. They have also been found to be effective in trapping free radicals up to 80%, reflecting their antioxidant activity (AOA) estimated through 2,2-diphenylpicrylhydrazyl (DPPH) free radical assay where the decreasing in absorbance of DPPH was considered a measure. These NPs were also taken for their binding potential with bovine serum albumin (BSA) and DNA, where they have shown up to 50% binding capacity due to trapping themselves into a grove of BSA and DNA. This feature enables them for medicinal purposes.

Monoclonal proliferation of B-cells with two integration sites of bovine leukemia virus proviral DNA in cattle with enzootic bovine leukosis.

The present study analyzed B-cell clonality and bovine leukemia virus (BLV) provirus integration sites in cattle with enzootic bovine leukosis (EBL) having BLV proviral copy numbers less or greater than the number of bovine nucleated cells. EBL cattle with BLV copy numbers less than the number of bovine nucleated cells showed monoclonal and biclonal proliferation of B-cells with one BLV provirus integration site. On the other hand, EBL cattle with BLV copy numbers greater than the number of bovine nucleated cells showed monoclonal proliferation of B-cells with two BLV provirus integration sites. These results suggest that superinfection of BLV can occur in EBL cattle.

Application of Real-time PCR with Mitochondrial D-Loop Specific Primers for Halal Authentication: Identifying Bovine Adulteration in Meatball Products

This study aimed to develop an analytical method using Real-time PCR and a specific primer to analyze bovine DNA in meatballs, addressing the issue of non-halal meat being used in halal meatballs. Real-time PCR enables rapid, specific, and sensitive detection, allowing for qualitative and quantitative identification of species in processed products. The research involved designing specific primers for bovine DNA using IDT software, followed by DNA isolation and testing for various parameters such as specifications, linearity, limit of detection, efficiency, and repeatability. The results demonstrated that the primer D-Loop 922 (forward: 5-ATTACCATGCCGCGTGAA-3', Reverse: 5'-GATGAGATGGCCCTGAAGAAA-3'), designed and tested in silico using Primer-BLAST software from NCBI, effectively identified bovine DNA in both fresh meat and meatballs at an optimum annealing temperature of 59.5°C. The real-time PCR method utilizing the D-loop 922 primer successfully amplified bovine DNA in both bovine samples and bovine meatballs at a minimum concentration of 1 ng, with coefficients of variation (CV) of 0.20% for bovine DNA and 0.22% for bovine meatballs. Consequently, the D-loop 922 primer met the testing criteria and can be utilized to authenticate the halal status of meatball products, supporting the implementation of Law No. 33 of 2014 regarding halal certification.

Prevalence of Bovine leukemia virus in Egyptian women’s breast cancer tissues

ABSTRACT Bovine leukemia virus (BLV) infects cattle around the world and presents in commercial bovine products. Several studies have suggested that BLV may play a role in breast oncogenesis. This study aimed to determine the risk factors for BLV infection and the presence of the BLV gene in breast tissue. A total of 100 formalin-fixed and paraffin-embedded breast tissue specimens were collected (50 confirmed cancer samples and 50 negative samples). DNA was extracted from breast tissues, and nested PCR was done to detect the tax gene of BLV. Immunohistochemical staining for ER, PR, Her2/neu, and Ki-67 was applied. Bovine leukemia virus DNA was detected in 16% of breast cancer tissue samples and in only 4% of tissue samples without cancer. No statistically significant association was reported between the presence of BLV and tumor grade, stage, or LN metastasis. A high percentage of positivity (100%, 88%, and 75%) was reported for ER, PR, and Ki-67 in BLV-positive cancer samples, respectively. There was a significant association between the BLV tax gene and breast cancer in Egyptian women. Thus, there is a need to control BLV transmission to humans from cattle, and further investigations about its risk related to public health are highly required.

Comprehensive diagnosis of abortion in cattle

Abstract. Abortion is one of the most common factors that has an extremely negative eff ect on the reproductive capacity of cattle, resulting in signifi cant economic losses. Abortions can be caused by infectious and non-infectious agents. Diagnosis of abortion is a diffi cult task, since only about 40% of abortions are diagnosed as infectious, and the remaining 60% - as unknown etiology. The goal was to establish the infectious etiology of abortion using complex diagnostics. The research was carried out in the laboratories of "Center of Veterinary Diagnostics" LLC (Kyiv). The selection of samples for research was carried out in the section hall of the LLC "Center of Veterinary Diagnostics" during the autopsy. The material for the study was a fetus and a vaginal smear from an aborted cow. Abortion occurred at 4 months of pregnancy. To establish the etiology, the following research methods were used: patho-anatomical, bacteriological, histological and molecular genetic (PCR-RT). Autolysis, light gray foci ≤ 2 mm in diameter in the lungs, and multiple light gray foci ≤ 1 mm in diameter in the liver were the main pathological fi ndings. The main pathohistological changes detected were focal myocarditis, intranuclear inclusion bodies in hepatocytes, multifocal coagulation necrosis in the lungs and liver, and diff use interstitial nephritis. Bacteriological culture was negative. DNA of infectious bovine rhinotracheitis virus (IBRV) and DNA of bovine herpesvirus type 4 (BHV-4) was isolated from the examined samples of fetal tissues and vaginal smear by PCR-RT. Using a comprehensive study, infectious agents and changes characteristic of the infectious process were identifi ed. On the basis of the obtained results, the etiology of the abortion was established as co-infection of IBRV and BHV-4. Complex diagnostics provides an opportunity to establish a cause-and-eff ect relationship between the identifi ed infectious agent and pathological changes in the fetus and the etiology of abortion. The results of a set of studies provide information on the manifestation of herpesvirus co-infection and complement the existing knowledge that can be used in the diagnosis of abortions in cows. Key words: abortion, cattle, IBRV, BHV-4, coinfection, PCR-RT, pathohistology, diagnosis.

Алгоритм определения количества ДНК провируса лейкоза крупного рогатого скота на основе ПЦР с флюоресцентной детекцией продуктов амплификации

Алгоритм определения количества ДНК провируса лейкоза крупного рогатого скота на основе ПЦР с флюоресцентной детекцией продуктов амплификации

Evaluation of commercial meat product food label conformity using multiplex PCR assay

This study evaluated the authenticity of pre-packaged processed beef products using multiplex PCR (mPCR) analysis on mtDNA. A total of 48 commercial beef products, including meatballs, burgers, corned beef, smoked beef, and sausages, were examined. They were categorized into three price groups: low (<5 USD/kg), medium (5–10 USD/kg), and high (>10 USD/kg). The DNA genome was extracted and used to amplify mtDNA 12S rRNA to identify bovine, porcine, dog, and rat ingredients, as well as mtDNA cytochrome c oxidase subunit 1 (COI) to detect bovine, chicken, and porcine ingredients. The results showed that all samples contained bovine DNA (155 bp), based on mPCR 12S rRNA. Moreover, chicken DNA was detected in 33 beef products (596 bp) based on mPCR COI. Mislabeling was detected in 56.25% of beef products. In addition, chicken DNA was detected in all three price groups. In conclusion, some products did not comply with the label composition labeling, and higher prices did not guarantee beef product originality.

A simplex and multiplex PCR assay for simultaneous detection of beef, pork, and chicken meat in sausages based on mitochondrial DNA Cytochrome oxidase sub-unit I

Adulteration in the processed beef product industry has long been an issue affecting consumers’ rights and legal protection as beef is an expensive raw material. The intense market competition opens up opportunities for manufacturers of processed beef products to counterfeit and mixes their products with other cheap meat ingredients such as chicken and pork. In addition, the use of pork to substitute beef in the production of sausages without proper labelling on the final products is both an adulteration and halal assurance issue in the Muslim-majority market. Multiplex PCR is a sensitive assay used for the identification of raw materials used in the production of processed food and it reduces the cost and time of analyzing a large number of samples. The purpose of this research was to apply the multiplex PCR assay targeting mitochondrial DNA Cytochrome Oxidase SubUnit I (COI) sequence and to determine the detection limits for DNA concentrations in beef, pork, and chicken. The DNA genomes that were extracted from each meat were diluted to concentrations of 25; 10; 1; 0.1; 0.01; and 0.001 ng/µL, and then used as a DNA template for the simplex- and multiplex-PCR. The results of simplex- and multiplex-PCR showed that the COI primers used were able to amplify the DNA of bovine, porcine, and chicken accurately as indicated by amplicons of 263 bp, 168 bp, and 596 bp, respectively. It was concluded that the simplex- and multiplex-PCR methods using COI primers can be used to identify bovine, porcine, and chicken DNA isolated from sausages with a sensitivity of 0.001 ng/μL.

Histamine derived Schiff bases and corresponding spinaceamines - synthesis, characterization and biological evaluation

The synthesis of eight new histamine-based Schiff bases and cyclic derivatives spinaceamines was accomplished by the reaction of corresponding aldehydes and histamine as amine-counterpart in absolute ethanol. Bearing in mind that the presence of the imino group/or spinaceamine core in the organic molecular scaffold could bring prominent pharmaceutical properties, all synthesised compounds have been screened for their antimicrobial activities against a range of different bacterial and fungal strains and antioxidant activity, as well as for interactions with bovine serum albumin (BSA) and DNA molecules. According to the observed MIC values, the best activity for both, fungal and bacterial species, was displayed by the compound 3c bearing histamine and quinoline moieties in the structure, especially for Staphylococcus aureus. Results obtained in the fluorescence quenching experiments have confirmed ability of tested compounds for DNA and BSA binding, while docking simulations revealed the site I of sub-domain IIA of BSA as a location of binding sites of tested compounds, as well as the minor grove and intercalation interactions with quite similar possibilities to be considered as compounds DNA-binding modes. Further, the molecular docking studies also gave insight into binding modes to the targeted enzymes (CYP51, cytochrome P450 and TyRS) related to the antifungal and antibacterial potential of tested compounds. Two compounds were selected, Schiff base 3c and spinaceamine 4d, to assess the stability of compounds under the physiological conditions (UV-vis spectroscopic study) and in silico ADME properties (SwissADME software), where both compounds have shown the stability after 72 hours in selected medium, and a potential as a drug based on their ADME properties.

Evaluation of the prevalence of bovine leukemia virus DNA in peripheral blood mononuclear cells of multiple sclerosis patients

Multiple sclerosis (MS) is an immune system-mediated neurodegenerative disease. Recent studies suggest that viral agents, especially the Epstein Barr virus (EBV), are etiological agents for MS. The roles of other viruses in MS have been investigated. Studies have shown an increase in the level of antibodies against bovine leukemia virus (BLV) in patients with MS. In this regard, our study aimed to examine the presence of BLV DNA in peripheral blood mononuclear cells (PBMCs) of MS patients in Iran. In this cross-sectional study, the presence of BLV in 109 Iranian MS patients and 60 healthy controls was evaluated. The isolated PBMCs were used for DNA extraction and PCR, using specific primers for two distinct genes. The mean age of the participants was 39 ± 9.5 years, and 27 (24.77%) of them were male. Clinical evaluation of these patients showed the most frequent MS type to be relapsing-remitting MS (RRMS) (71; 65.14%). BLV evaluation did not show any BLV DNA presence in the PBMCs of individuals in either the MS or healthy control groups. Therefore, our study showed no evidence of BLV infection in Iranian MS patients.

Multiplex PCR for simultaneous detection of DNA contamination from nonhalal species in beef products

A large number of mixtures of non-halal ingredients such as pork and rat meat in processed foods has worried the public, especially adherents of the Islamic religion. There is a need for a method to detect the presence of non-halal contaminants in several foods found in the community. This study aims to obtain a valid method by proposing multiplex PCR to detect DNA in processed meat foods. The sample of this research was processed beef (meatballs) obtained from five parts of the region from Surabaya, Indonesia. Multiplex PCR results on target species (pigs, mice, cattle) showed thick and clear DNA bands. This indicates that the amplification runs optimally. The designed primer can be used to test samples of processed meat foods on the market. The results showed that there was no adulteration with rat meat or pork in all samples in all areas in Surabaya. The results of electrophoresis showed that there was only one DNA band measuring 495 bp which was the result of the amplification of bovine DNA. The multiplex PCR developed in this study proved to be effective for detecting non-halal contaminants in meat-processed food products to be used as a reference for other tests.

Adulteration Test of Chicken DNA (Gallus gallus) by the Multiplex PCR Method Using a Specific Primer for Mitochondrial DNA CO1

Food adulteration cases continue to increase in line with the increasing public need for food. Multiplex PCR is one type of PCR method that is often used to detect adulteration in food. This study aims to confirm the performance of primers that have been designed based on the CO1 gene that specifically amplifies chicken DNA by a primer specificity and sensitivity test using single and multiplex PCR method. The results showed that the primer had specific properties because it is capable of amplifying the target DNA according to its size. The sensitivity test showed that the CO1 primers for chicken have a sensitivity of up to 10^-3 ng/µl similar to the pig's D-loop primers, while the CO1 primers for horses have a sensitivity of up to 10^-2 ng/ l similar to the Cyt b for dogs. Sampling test using five types of meatballs by the multiplex PCR method showed that the samples detected animal DNA that matched the respective raw materials for making it, while sampling using commercial meatballs showed that only three samples contained bovine DNA and it could be concluded that the other two samples had been adulterated with chicken meat.

Detection of genetic material of causative agents of animal viral diseases in blood-sucking dipterans from the Tyumen Region

The purpose of the research is to test blood-sucking dipterans collected in the Tyumen Region for genetic material of viruses that cause dangerous diseases in live-stock animals.Materials and methods. From May to October 2021, blood-sucking insects of the Diptera order whose species membership was established by tabular keys were collected on pastures and farms in ten Tyumen Region districts. In 60 samples formed from the captured insects according to the taxonomic affiliation and the period and place of collection, the presence of genetic material of the leukemia provirus and the dermatitis nodularis virus of cattle (bovine) and the African swine fever virus was evaluated by polymerase chain reaction (PCR) in real time.Results and discussion. Adult insects collected for the PCR analysis were blood-sucking flies (family Muscidae, genus Stomoxys), mosquitoes (family Culicidae, genus Aedes), midges (family Simuliidae, genera Byssodon and Schoenbaueria), horseflies (family Tabanidae, genera Hybomitra, Tabanus and Haematopota), and biting midges (family Ceratopogonidae, genus Culicoides). As a result of the PCR testing of the samples for the bovine leukemia provirus DNA, 1 out of 13 samples of Stomoxys spp. (7.7%) and 1 of 13 samples of Hybomitra spp. (7.7%) were positive. The bovine leukemia provirus DNA detected in blood-sucking insects indicates the presence of this pathogen in the insect collection area as well as their possible involvement in its spread. Further research is needed on the Stomoxys spp. and Hybomitra spp. vector competence in vivo, considering natural and climatic features of the Tyumen Region.

Structural characterization of protein-DNA complexes using small angle X-ray scattering (SAXS) with contrast variation.

Molecular and atomic level characterization of transcription factor (TF)-DNA complexes is critical for understanding DNA-binding specificity and potentially structural changes that may occur in protein and/or DNA upon complex formation. Often TFs are large, multidomain proteins or contain disordered regions that contribute to DNA recognition and/or binding affinity but are difficult to structurally characterize due to their high molecular weight and intrinsic flexibility. This results in challenges to obtaining high resolution structural information using Nuclear Magnetic Resonance (NMR) spectroscopy due to the relatively large size of the protein-DNA complexes of interest or macromolecular crystallography due to the difficulty in obtaining crystals of flexible proteins. Small angle X-ray scattering (SAXS) offers a complementary method to NMR and X-ray crystallography that allows for low-resolution structural characterization of protein, DNA, and protein-DNA complexes in solution over a greater size range and irrespective of interdomain flexibility and/or disordered regions. One important caveat to SAXS data interpretation, however, has been the inability to distinguish between scattering coming from the protein versus DNA component of the complex of interest. Here, we present a protocol using contrast variation via increasing sucrose concentrations to distinguish between protein and DNA using the model protein bovine serum albumin (BSA) and DNA and the LUX ARRYTHMO TF-DNA complex. Examination of the scattering curves of the components individually and in combination with contrast variation allows the differentiation of protein and DNA density in the derived models. This protocol is designed for use on high flux SAXS beamlines with temperature-controlled sample storage and sample exposure units.