What is Supershift in EMSA?
What is Supershift in EMSA?
The EMSA supershift is a EMSA experiment carried out using a third lane loaded with the radiolabeled nucleic acid, a protein mixture and an antibody for a specific protein. If an extra retardation is observed, this is due to the formation of a larger complex including the antibody.
What are the steps of EMSA?
The gel shift assay consists of three key steps: (1) binding reactions, (2) electrophoresis, (3) probe detection. The order of component addition for the binding reaction is often critical.
How do EMSA assays work?
EMSA is a widely used technique to study protein–DNA or protein–RNA interactions. This technique relies on the principle that protein–nucleic acid complexes migrate slower relative to unbound nucleic acid during electrophoresis in a nondenaturing polyacrylamide (PAGE) or agarose gel.
How EMSA can be used in protein-DNA binding studies?
The electrophoretic mobility shift assay (EMSA), one of the most sensitive methods for studying the DNA-binding properties of a protein, can be used to deduce the binding parameters and relative affinities of a protein for one or more DNA sites or for comparing the affinities of different proteins for the same sites1.
What is cold probe in EMSA?
Usually, a specific competitive probe is unlabeled (referred as cold probe) and has the same sequence (or at least with the same binding domain) as that of labeled probes. The mutant probe is also unlabeled, and has the sequence as that of labeled probes, but the binding domain has been mutant with one or more bases.
What controls are used in EMSA?
EMSA Controls
| Types of controls | Description | Pack |
|---|---|---|
| EMSA-P/N Ctrl | EMSA Positive/Negative | 10 loadings/ea |
| EMSA-P/ Ctrl | EMSA Positive controls | 10 loadings |
| EMSA-N/ Ctrl | EMSA Negative controls | 10 loadings |
Why is ChIP preferred over EMSA?
In contrast, the chromatin immunoprecipitation (ChIP) offers a distinct advantage over EMSA and in vivo footprinting, since the ChIP technique not only specifies which nucleotides are bound, but also identifies the interacting protein(s) in the context of in vivo samples [7].
Is vivo a ChIP?
Chromatin immunoprecipitation, or ChIP, refers to a procedure used to determine whether a given protein binds to or is localized to a specific DNA sequence in vivo. The diagram below illustrates the basic steps of this procedure. DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo.
What is ChIP PCR?
ChIP-PCR is performed to analyze histone modifications and/or protein binding to a known subset of target loci in the genome. In ChIP-PCR, immune-enriched DNA fragments are identified and quantified using widely available PCR or qPCR reagents and technologies.
