The Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit provides a method for studying RNA-protein interactions using electrophoretic mobility shift assays (EMSA). A non-radioactive solution.

LightShift Chemiluminescent RNA EMSA Kit Features:

•Sensitive — Chemiluminescent detection is comparable to radioactive detection
•Saving time b> — X-ray film can be developed after 1 to 5 minutes of exposure compared to the 16 hours required for radioactive systems
•Flexible — Compatible with RNA biotinylated by different methods Probes
•Easy to use — Complete kit includes optimized reagents for binding reactions and RNA probe detection
•Radioactive-free — No radioactive RNA is present The waste problem of probes

RNA EMSA kit uses biotinylated RNA probes and chemiluminescent substrate systems to quickly and safely detect RNA-protein complexes with the same sensitivity as traditional ³²P-isotope methods are comparable. The complete kit contains all reagents needed to construct and optimize protein-RNA binding conditions, positive controls for protein-RNA interactions, and chemiluminescent detection of nucleic acid interactions.

About LightShift Chemiluminescent RNA EMSA Kit
LightShift Chemiluminescent RNA EMSA Kit detects changes in gel electrophoretic migration patterns similar to commonly used DNA gel migration experiments. In vitro techniques for protein-RNA interactions. In RNA EMSA, labeled RNA probes are incubated with protein samples to initiate binding. After complex formation, samples were separated by nondenaturing polyacrylamide gel electrophoresis. Because RNA-protein complexes migrate more slowly than free RNA probes, migration distances can be observed using RNA gel shift experiments. The specificity of the RNA-protein interaction was verified by competing with excess unlabeled RNA binding to reduce the specific interaction signal. In general, mutated or unrelated RNA probes are not expected to compete for specific interactions and the intensity of specific band migration should not be reduced when detected in EMSA. The complete LightShift Chemiluminescent RNA EMSA Kit contains all the reagents needed to set up and optimize RNA gel shift assays, including positive controls for RNA-protein complex formation.

LightShift Chemiluminescent RNA EMSA Kit uses biotinylated RNA probes, horseradish peroxidase-labeled streptavidin, and chemiluminescent detection to provide detection similar to that achieved with radioactive RNA probes. sensitivity, but faster detection. Labeled RNA probes can be purchased commercially or generated by a runawayin vitro transcription reaction using biotinylated nucleotides or by adding biotin using the Thermo Scientific Pierce RNA 3' Terminal Biotinylation Kit. The label is generated by enzymatic attachment to the 3' end of the RNA chain. The LightShift Chemiluminescent RNA EMSA Kit is effective for biotinylated RNA probes by any of these three methods; however, during runawayin vitrotranscribed RNA probe synthesis, RNA II The hierarchical structure may be affected by internal incorporation of biotinylated nucleotides. Therefore, for some interactions, custom synthetic RNA probes or 3' end biotinylated probes may be required to achieve appropriate protein-RNA interactions.

About the Positive Control for the LightShift Chemiluminescent RNA EMSA Kit
The positive control included in the LightShift Chemiluminescent RNA EMSA Kit is the Iron Responsive Element (IRE) RNA Probe. Needle. The IRE binding reaction is set up and detected simultaneously with other experimental samples. Under iron-deficient conditions, iron-responsive protein (IRP) remains bound to IRE RNA present in the cell, effectively inhibiting the translation of ferritin (an iron storage protein) and then inhibiting the transferrin receptor. Under iron-rich conditions, IRE binding activity is lost and ferritin and transferrin translation proceeds. This system is ubiquitous and produces robust band migration. Incubating the positive control reaction with a 200-fold molar excess of unlabeled IRE RNA will reduce the specific IRE band migration signal by approximately 70%, whereas a similar fold excess of irrelevant RNA probe will not significantly reduce IRE band migration. These controls can be used in each RNA gel shift experiment to verify correct setup, electrophoresis, transfer, and detection of protein-RNA complex formation.

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