Pierce™ Magnetic RNA-Protein Pull-Down Kit - FAQs

View additional product information for Pierce™ Magnetic RNA-Protein Pull-Down Kit - FAQs (20164)

11 product FAQs found

Can I use Coomassie or silver staining to detect proteins isolated with the Pierce Magnetic RNA-Protein Pull-Down Kit (Cat. No. 20164)?

Proteins isolated with the Magnetic RNA-Protein Pull-Down Kit cannot be detected by Coomassie or silver staining. Gels can be used for detection depending on the amount of total protein used in the experiment. If the protein of interest has an antibody available, it can usually be detected via western blotting. Finally, if the protein isolated is unknown, it can be analyzed by mass spectrometry.

After using the Magnetic RNA-Protein Pull-Down Kit, I would like to analyze my elution fraction by mass spectrometry (MS). How can I do this?

The elution fraction is compatible with preparation of peptides. Samples may be processed using the Mass Spec Sample Prep Kit for Cultured Cells (Cat. No. 89840). Alternatively, the elution fraction may be separated by denaturing PAGE. Bands of interest can be excised, and digested using the In-Gel Tryptic Digestion Kit (Cat. No. 89871).

I am using the Magnetic RNA-Protein Pull-Down Kit and am getting a low signal on my western blot. What is the problem?

Here are the possible causes and solutions:

- Insufficient signal: Increase amount of secondary antibody; Use a more sensitive chemiluminescent detection (e.g., SuperSignal Dura or SuperSignal Femto Chemiluminescent Substrate).
- Poor antibody quality: Pre-screen antibody with cell lysate; Include cell lysate as a control on western blot.
- Protein was insufficient in lysate: Increase amount of sample; Identify alternate source of protein; Use a more concentrated lysate.

With the Magnetic RNA-Protein Pull-Down Kit, I am getting high non-specific binding of the RNA-binding protein. Why is this?

Here are possible causes and solutions:

- Binding reaction was not optimized: Optimize incubation time, temperature, salt and detergent for binding reactions.
- Insufficient washing stringency: Increase stringency of wash buffer; add salt and/or detergent.
- Ratio of labeled RNA to lysate was not optimized: Titrate labeled RNA with protein lysate; Reduce the concentration of lysate to ~2mg/mL.

I got no recovery of the RNA-binding protein when I used the Magnetic RNA-Protein Pull-down Kit. What should I do?

Here are possible causes and solutions:

- Insufficient amount of target protein in the sample: Increase amount of sample.
- Sample was not compatible with the binding reaction: Buffer exchange sample using Zeba Desalting Columns.
- Binding reaction was not optimized: Optimize incubation time, temperature, salt and detergent for binding reactions; Titrate amount of labeled RNA to protein lysate; Use a more concentrated lysate.
- RNA binding protein had low affinity for labeled RNA: Add crosslinking reagent (e.g., UV, etc.) after protein has bound RNA.

With the Magnetic RNA-Protein Pull-down Kit, I got inefficient recovery of the RNA-binding protein. What could have happened?

Here are possible causes and solutions:

- Binding reaction was not optimized: Optimize incubation time, temperature, salt and detergent for binding reactions; Titrate amount of labeled RNA to protein lysate; Use a more concentrated lysate.
- Insufficient amount of magnetic beads used for capture: Increase amount of magnetic beads for capture.
- Insufficient amount of RNA used for capture: Increase amount of labeled RNA in reaction; Confirm good ligation efficiency.

With the Magnetic RNA-Protein Pull-down Kit, my labeling efficiency was low. How can I improve the labeling efficiency?

Ensure that the control reaction in the kit is run to confirm that the labeling reaction is efficient. If the control RNA labels well, but the test RNA does not, optimization is required. Addition of DMSO or heat may help to relax the structure. Additionally, ensure that there are no traces of organic reagent in the reaction (if RNA was extracted before labeling). Ensure that 70% ethanol wash was utilized after precipitation. Ensure that the RNA concentration is sufficient for the labeling reaction.

What is the maximum length of RNA that I can use with the Magnetic RNA-Protein Pull-down Kit?

RNA up to 450 nucleotides has been tested, but with optimization, labeling of longer RNA may be achievable. The RNA must have an accessible 3’-OH for the ligation reaction. 

How can I prevent RNA degradation in the Magnetic RNA-Protein Pull-Down Assay?

Work in a clean nuclease-free environment.

  • Ensure that all plastics are nuclease-free. 
  • After in vitro transcription, check RNA integrity by gel electrophoresis.

    • How do I prepare my labeled RNA probe for my RNA Protein Pull-Down Assay?

    Labeled RNA probes can be purchased commercially or generated through either run-off in vitro transcription reactions with biotinylated nucleotides or through enzymatic ligation of biotin tags to the 3' terminus of an RNA strand using the RNA 3' End Biotinylation Kit (Cat. No. 20160) or RNA 3’ End Desthiobiotinylation Kit (Cat. No. 20163). Desthiobiotinylated cytidine is utilized for the pulldown assay to enable a gentle elution to capture protein-RNA complexes. Reagents and conditions are included in the kit to relax the RNA for better labeling efficiency. Additionally, suggestions are included in the instruction manual for optimization of the ligation reaction. An RNA control is included in the kit to assess kit labeling efficiency.

    How does the Magnetic RNA-Protein Pull-Down Assay (Cat. No. 20164) work?

    The RNA Protein Pull-Down Assay is an in vitro technique for detection of protein-RNA interactions using labeled RNA as bait for protein. RNA is labeled with desthiobiotinylated cytidine and bound to streptavidin magnetic beads. The magnetic beads are then incubated with cell lysate to capture proteins that interact with the attached RNA Proteins are then gently eluted using biotin and detected by western or MS analysis. Because of the gentle elution (through use of desthiobiotin), RNA protein complexes may be captured and detected. The kit contains a pull-down control (RNA and antibody) that capture the correct protein from most lysates.