Smart-circ-Pull™ circRNA Pull-down Kit

The Generation and Function of CircRNA

CircRNAs localized in the nucleus can function as regulatory molecules for their host gene transcription by interacting with the U1 small nuclear ribonucleoprotein (U1-snRNP) and enhancing the activity of the RNA polymerase II (Pol-II) complex, or by recruiting the methylcytosine dioxygenase TET1 to the promoter region (a). When exported to the cytoplasm, circRNAs can act as sponges or decoys for microRNAs and RNA-binding proteins (RBPs), or modulate the half-life of specific RBPs, either opposing (red T line) or facilitating their proteasome-mediated degradation (green arrow) (b, c). CircRNAs have also been shown to function as protein scaffolds (c). By promoting the co-localization of enzymes and their substrates, they can enhance reaction kinetics (yellow arrow). Finally, circRNAs containing internal ribosome entry site (IRES) elements and AUG sites (green line) can be translated via a cap-independent mechanism (dashed arrow; red line indicates stop codon). The latter is facilitated by the presence of methyladenosine (m6A) and the involvement of reader proteins YTHDF3 and IRES-specific translation initiation factor eIF4G2 (d). The protein isoforms translated from circRNAs will share partial primary sequences with linearly encoded proteins, while the remaining portions of the polypeptide are unique to the isoforms encoded by circRNAs.

Overview

Description

Smart-circ-Pull™ circRNA Pull-down Kit (circRNA Protein Pull-down)

Properties

Components

Components of circRNA Pull-down Kit
 
 

ITEM	NAME	SIZE		NOTE
The following components need to be stored at 4 ° C upon receipt.
IF9304	Streptavidin Magnetic Beads Note: The surface of Streptavidin magnetic beads has been pre-coated with nucleic acid oligo, which can minimize non-specific binding to the probe	1 mL	+4 °C	Magnetic beads should not be frozen or dried. Do not centrifuge at high speed
R-pull-002	1×circRNA Immobilized Buffer	5 mL	+4 °C
R-pull-003	1×Magnetic Beads Wash Buffer	20 mL	+4 °C
R-pull-004	10×Protein-circRNA Binding Buffer	1 mL	+4 °C
R-pull-005	R-pull 1×Wash Buffer	15 mL	+4 °C
R-pull-006	R-pull Elution Buffer (Denaturing)	1.5 mL	+4 °C
R-pull-007	R-pull Elution Buffer  (non-denaturing, solution A)	4 mL	+4 °C
R-pull-008	R-pull Elution Buffer  (non-denaturing, solution B)	0.5 mL	+4 °C
The following components need to be stored at RT upon receipt.
IF9217	QuickStain™ One-Step Protein Gel (PAGE) Blue Stain	50 mL	RT
The following components need to be stored at -20 ° C upon receipt.
R-pull-010	DNase I(Enzyme and 10×Buffer)	100 U	-20 °C	Used for the removal of DNA from cell lysate
R-pull-011	RNase Inhibitor	50 μL	-20 °C
R-pull-012	tRNA (10 mg/mL)	50 μL	-20 °C
R-pull-013	50% Glycerol	500 μL	-20 °C
R-pull-014	R-pull Loading Buffer(5×)	1 mL	-20 °C	For SDS-PAGE
AT0098	Goat anti mouse Conjugated HRP	100 µL	-20 °C	For WB
AT0097	Goat anti rabbit Conjugated HRP	100 µL	-20 °C	For WB

 

Applications

Application Image

Highlights

1. This kit is suitable for adherent cells, suspension cells, animal tissues, and plant tissues, and can be used with endogenous, overexpressed, purified recombinant proteins, or proteins translated in vitro as sample types. It is applicable to biotin-labeled circular RNA probes of various lengths and complexities.

 

2. This kit directly captures the target protein using biotin-labeled circular RNA probes. Antibodies and centrifugation are not required during pull-down. After the RNA labeling reaction, only minimal manual operation time is needed (less than 3 hours).

 

3. This kit is suitable for detecting the interaction between RBP and circRNA, downstream validation via Western blot for known proteins, and mass spectrometry identification for unknown proteins.

 

4. This manual serves as a comprehensive technical guide for circRNA Pull-down experiments. After reading this step-by-step guide, all difficulties will be effortlessly resolved, and RNA Pull-down will be fully within your grasp.

Work Flow Image