DNA/RNA Pull Down Service

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DNA/RNA Pull Down Service

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CD BioSciences provides DNA/RNA pull-down services for screening plant proteins (such as transcription factors) interacting with target nucleic acid fragments. By combining with MS, interacting proteins can be qualified simultaneously, further revealing gene regulatory mechanisms and transcriptional regulatory networks.

Nucleic acid-protein interactions are central to cellular functions, including essential metabolic processes in life activities such as protein synthesis and mRNA assembly, and the study of nucleic acid-protein interactions is a fundamental life-science tool. In recent years, pull-down is one of the main experimental techniques for screening and detecting RNA/DNA-interacting proteins.

Workflow of DNA/RNA pull-down assay. Figure 1. Workflow of DNA/RNA pull-down assay. (Cozzolino, F., et al., 2021)

Service Content

Workflows

Our full array of services covers every step of the DNA/RNA pull-down, including

  • Target RNA preparation.

    • a) Construction of RNA overexpression plasmid, gene synthesis, and sequencing verification.

    b) In vitro transcription template preparation, design primers containing T7 promoter, PCR amplification to obtain transcription template.

    c) In vitro transcription, uses PCR product as a template to get target RNA.

  • Target DNA preparation.

Design and direct synthesis of specific DNA probes for target regions.

  • Desulfurized biotin-labeled probes.
  • Magnetic bead enrichment of DNA/RNA.
  • Nucleus extracts incubated with magnetic beads-DNA/RNA probes.
  • Binding protein elution, and silver staining for quality control.
  • Mass Spectrometry (MS) or Western Blot to identify proteins.

Optimization service of DNA/RNA pull-down

  • Reduces nonspecific binding

    • a) Optimization of probe design.

    b) Optimize conditions such as incubation time, temperature, and salt concentration.

    c) Selection of buffer systems with high rigor.

    d) Screening for appropriate probe-to-sample ratio.

  • Protein concentration screening

We try different concentrations of proteins to determine the optimal concentration range for the strongest signal. In addition, we use standards or internal controls for quantitative analysis to correct for variations in signal.

  • Improve sample purity

We help clients optimize sample processing steps, such as using high-purity nucleic acid extraction methods to reduce impurities. In addition, we can also use specific antibodies for pre-treatment.

Sample Requirements

Sample type Requirements Transport Details
Cells > 5×107 Dry ice transport Suspension cells are centrifuged at room temperature (1000-1500rpm, 5min). The cells are snap-frozen in liquid nitrogen for 15min and stored at -80℃.
Tissue > 2g Quickly cut plant tissue into small pieces (within 1min), about 200mg, freeze in liquid nitrogen for 15min, and store at -80℃.
Nucleoprotein > 0.5mg/mL Nucleoprotein > 300ug.

Application Fields

  • Study the binding of transcription factors to gene promoters to reveal gene regulatory mechanisms and transcriptional regulatory networks.
  • Research on the interaction between critical proteins and nucleic acids in signaling pathways, such as activators and repressors.
  • Screening for small molecule compounds or drugs that bind to specific nucleic acid sequences, searching for new drug targets, and developing potential drug therapies.
  • Compare nucleic acid-protein interactions in different species, tissues, or under different conditions to help understand changes in genome evolution, tissue specificity, and environmental adaptation.

CD BioSciences is dedicated to plant protein research, with cutting-edge facilities and optimized protocols to ensure effective screening and enrichment of plant proteins interacting with DNA/RNA, capable of identifying and characterizing binding partners, regulatory elements, and functional regions. If you have any questions, please contact us and we will customize our services to meet your specific needs.

References

  1. Cozzolino, F., et al. (2021). Protein–DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era. Journal of Proteome Research. 20(6), 3018-3030.
  2. Sui, H., et al. (2020). A pull-down assay using DNA/RNA-conjugated beads with a customized competition strategy: An effective approach to identify DNA/RNA binding proteins. MethodsX. 7, 100890.

For research use only, not for clinical use.