Protein-RNA interactions play important roles in the cell including structural, catalytic, and regulatory functions. Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) can be used to detect the association of individual proteins with specific nucleic acid regions. RIP is a powerful technique used to detect the association of individual proteins with specific RNA molecules in vivo. Live cells are treated with highly reactive, reversible crosslinker formaldehyde to generate protein-RNA cross-links between molecules that are in close proximity in vivo. Following immunoprecipitation of a protein of interest and cross-link reversal, associated RNAs can be recovered, characterized, and quantitated by reverse transcriptase polymerase chain reaction (RT-PCR).
The Sigma Life Science RIP kit provides all reagents needed for successful RIP when paired with appropriate antibody. The kit includes two lysis buffers, one mild for less tightly bound RNPs, the other harsh for strongly associated RNPs. Protein A magnetic beads are provided to collect immune precipitates and facilitate washing. The kit also includes both rabbit and mouse IgG for negative control RIPs, a rabbit anti-mouse bridging antibody to bind mouse monoclonal primary antibodies efficiently to protein A on the magnetic beads, as well as both protease and ribonuclease inhibitors to protect the RNPs from degradation during RIP.
Features and Benefits
Workflow
Application Data
Figure 1
RIP Lysate prepared from HeLa cells (0.75 x 106 cell equivalents per IP) were immunoprecipitated using 1 µg of either a normal Rabbit IgG, or Anti-SNRP70 antibody and the Imprint RNA Immunoprecipitation Kit. Immunoprecipitation of SNRP70-associated RNA was validated by qPCR using Control Primers, U1 snRNA.
Figure 2
RIP Lysate prepared from HeLa cells (0.75 x 106 cell equivalents per IP) were immunoprecipitated using 1 µg of either a normal mouse IgG, or Anti-PABPC1 antibody along with Anti-mouse antibody produced in Rabbit and the Imprint RNA Immunoprecipitation Kit. Immunoprecipitation of PABP-associated RNA was validated by qPCR using Control Primers, Actin B.
Figure 3
RIP Lysate prepared from HeLa cells (2.0 x 106 cell equivalents per IP) were immunoprecipitated using 1 µg of either a normal Rabbit IgG, or Anti-HUR antibody and the Imprint RNA Immunoprecipitation Kit. Immunoprecipitation of HUR-associated RNA was validated by qPCR using Control Primers, Actin B.
Figure 4
RIP Lysate prepared from HeLa cells (0.5 x106 or 1.7 x 106 cell equivalents per IP) were immunoprecipated using 2.5 μg of either a normal Rabbit IgG or Anti-HUR antibody and the Imprint RNA Immunoprecipitation Kit. Immunoprecipitation of HUR-target and non-target associated RNA was validated by qPCR using Control Primers Actin B and JUN.
RIP-qRT-PCR: Data Analysis Calculation Shell (30 Kb)
RIP Kit Components and Storage
The RIP kits are shipped at two different temperatures. The wet ice shipment contains Part 1. The dry ice shipment contains Part 2. Storage temperatures are indicated below.
| Imprint RNA Immunoprecipitation Kit Part 1 – catalog number RIPPART1 |
2-8°C | |
| B0314 | Mild Lysis Buffer | 1 x 3 mL |
| B0439 | Harsh Lysis Buffer | 1 x 3 mL |
| B0564 | RIP Wash Buffer | 2 x 75 mL |
| B0689 | Protein A Magnetic Beads* | 1 x 300 µL |
| I5381 | IgG from mouse serum | 1 x 1 mg |
| I5006 | IgG from rabbit serum | 1 x 1 mg |
| Imprint RNA Immunoprecipitation Kit Part 2 - catalog number RIPPART2 |
-20°C | |
| R1158 | Ribonuclease Inhibitor | 1 x 2.5 KU |
| P8340 | Protease Inhibitor Cocktail | 2 x 125 µL |
| M7023 | Anti-mouse IgG antibody produced in rabbit | 1 x 60 µL |
Order Information
| Product No. | Description | Quantity | Add to Cart |
| RIP-12Rxn | ImprintR RNA immunoprecipitation kit (with negative control) | 12 Reactions |  |