Epigenetics

Imprint® RNA Immunoprecipitation Kit

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

  • Xtra Protein A magnetic beads with higher binding capacity
  • Step-by-step protocols for cell lysis and immunoprecipitation
  • RNAse inhibitors and RNAse-free reagents to maintain RNA integrity
  • Negative control and antibody included

 

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