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Cat. No.: RIPAK-22 (for 22T)

Description

RNA Immunoprecipitation Assay Kit with Protein A/G Agarose, also known as RNA-binding protein immunoprecipitation assay or RIP assay kit, is used to precipitate RNA fragments bound to target proteins through immunoprecipitation, which are then detected by methods such as PCR, qPCR, or NGS.

Transcriptional regulation is crucial for gene expression in eukaryotes, but mRNA levels do not always directly correlate with protein levels, partly due to post-transcriptional regulation of mRNA. Key to post-transcriptional regulation is the interaction between RNA-binding proteins (RBPs) and their target mRNAs. RBPs influence mRNA localization, modification, stability, and translation levels by forming ribonucleoprotein (RNP) complexes with mRNA targets. With the evolution of prokaryotes to eukaryotes and the development of the nuclear membrane, the number of RBPs has significantly increased, focusing research on post-transcriptional gene expression on RBPs. It's crucial to identify unknown mRNA targets from RNP complexes to understand the mechanisms and functions of RBPs and their impact on protein expression levels. Additionally, RNA-binding proteins are not limited to mRNA but also include various non-coding RNAs such as long non-coding RNAs (lncRNAs) and small RNAs (sRNAs). Chromatin Immunoprecipitation (ChIP) is used to detect DNA targets bound by DNA-binding proteins in cells. Similarly, RNA Immunoprecipitation (RIP) is used to detect RNA targets bound by RNA-binding proteins in cells. Through immunoprecipitation of RNP complexes with specific antibodies against RBPs, RNA purified from the RNP complexes is then detected by methods such as qRT-PCR or high-throughput sequencing. RIP detection helps understand protein-RNA interactions and explores the molecular mechanisms underlying post-transcriptional gene expression regulation.

The RIP Assay Kit (Protein A/G Agarose) employs Protein A/G Agarose, which is suitable for immunoprecipitating a wider range of antibodies compared to Protein A Agarose or Protein G Agarose. This includes mouse IgG1, IgG2a, IgG2b, IgG3, IgA, rat IgG1, IgG2a, IgG2b, IgG2c, rabbit IgG, rabbit and goat polyclonal Abs, as well as human IgG1, IgG2, IgG3, and IgG4.

The RIP Assay Kit (Protein A/G Agarose) provides all the necessary reagents for RIP experiments. The Protein A/G Agarose beads for RIP are covalently coupled to 4% cross-linked, fast-flow agarose, where each milliliter of Protein A/G agarose beads can bind over 25 mg of human IgG. Proteinase and ribonuclease inhibitors, including PMSF and RNase Inhibitor, are provided to protect RNP complexes from degradation during immunoprecipitation. RNA purification kits, RNA reverse transcription kits, and RNA quantitative detection kits can be purchased separately from us.

If used for standard RNA immunoprecipitation, this assay kit can immunoprecipitate up to 22 samples.

Components

• Protein A/G Agarose
• Lysis Buffer
• NT2 Wash Buffer
• Elution Buffer
• RNase Inhibitor
• DTT
• PMSF

Precautions

• Strict measures must be taken to ensure no RNase contamination during the operation. Please wear masks and gloves to prevent contamination of samples by RNase on the surface of the body as much as possible. Avoid breathing or speaking directly towards the samples, reagent kits, or consumables to prevent RNase contamination.

• All reagents and consumables used must be RNase-free, and care should be taken to avoid contamination. If consumables may be contaminated with RNase, consider soaking them overnight in 0.01% DEPC water, followed by autoclaving at high temperature and pressure and drying.

• To ensure the binding of RNA with specific target proteins, the samples used in RIP experiments should be freshly prepared as much as possible.

• Protein A/G Agarose must be thoroughly resuspended before use, i.e., shaken several times to ensure thorough mixing.

• To ensure that RNA and proteins are not degraded during RIP operations, the corresponding RNase Inhibitor and PMSF should be added at the indicated locations in the instructions. All steps should be performed on ice as much as possible to reduce possible RNase activity.

• When washing Protein A/G Agarose, be careful to remove the supernatant gently. It is better to leave a small amount of supernatant than to remove Protein A/G Agarose. If the background is high, increase the number of washes.

• Antibodies used for RIP detection can be verified by Western blotting to determine if they can be applied to IP detection before conducting RIP experiments.

• This assay kit does not provide negative control antibodies IgG. It is necessary to separately order IgG corresponding to the species of the specific target protein antibody from us.

• This product is for scientific research use by professionals only. It is not intended for clinical diagnostics or treatments, food, or pharmaceuticals, and should not be stored in ordinary residences.

• For your safety and health, please wear lab coats and disposable gloves during operation.

Storage

Protein A/G Agarose should be stored at 4°C, while the other components should be stored at -20°C, both remaining effective for at least one year. Lysis Buffer and NT2 Wash Buffer can also be stored at 4°C and remain effective for at least one month.

Only for research and not intended for treatment of humans or animals