Whole cell lysate RIP troubleshooting tips

Step Potential Problems Experimental Suggestions
Immuno- precipitation Antibody doesn’t immunoprecipitate protein in the RIP lysate
  • Confirm the antibody can immuneprecipitate the RBP of interest by IP Western prior to RIP analysis
  • Choose an antibody directed to a different epitope of the antigen.
  • Use Milliporesigma’s RIPAb+ validated antibodies where possible
  • Perform IP from a dilution series of antibody with a fixed amount of RIP lysate or vice versa.
  • Increase incubation time of the antibody of interest with the RIP lysate to overnight at 4°C.
  • Confirm antibody isotype is compatible with immunoprecipitation by Protein A or G. This kit is not recommended for use of IgM or chicken IgY antibodies.
Insufficient quantity of magnetic beads in immunoprecipitation
  • The magnetic beads settle to the bottom of the tube over time. Make sure the magnetic beads are well mixed prior to removing the appropriate volume for IP.
  • Carefully aspirate beads when using vacuum aspirator and use a high strength neodymium magnetic rack such as the MagnaGrIP Rack (Cat. #: 20-400).
RNA Purification Incomplete Proteinase K digestion
  • When performing proteinase K digestion, make sure that the temperature is set at around 55°C. Proteinase K will be inactivated by prolonged incubation at temperatures above 65°C.
Poor A260/A280 ratios
  • Avoid the interphase when extracting RNA using phenol:chloroform and choloroform extractions.
  • Assess presence of RNAses by evaluating RNA extracted from lysate inputs on a Bioanalyzer or Experion instrument.
Low RNA yield
  • Most RNA-binding protein immunoprecipitations do not yield measureable amounts of RNA. Sub nanogram quantities of RNAs can however be detected by RT- PCR.
  • If RNAs are not detectable following cDNA synthesis, consider Immunoprecipitation troubleshooting above.

RNA degraded
  • Use RNAse inhibitor in solutions as recommended in this protocol.  Make certain RNAse free work conditions exist and RNAses are not being introduced.
  • Follow the guideline for the RNase control at the beginning of the protocol
  • Use RNAse inactivating reagents to ensure work area and materials are RNAse free
No RNA detected
  • Increase incubation time for the ethanol precipitation at -80°C.
  • The RNA ethanol precipitates are sometimes very small. Be sure not to suck up RNA precipitate when removing the supernatant.
  • Confirm the antibody can immunoprecipitate the RBP of interest by IP Western prior to RIP analysis.

For more information about RIP experiment guides, troubleshooting tips and supplementary protocols, please view our RNA Immunoprecipitation (RIP) page.

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