EMSA is a powerful tool for evaluating DNA-protein or RNA-protein interactions. The standard techniques used in EMSA involve the use of radioactive oligonucleotide probes labeled with radioisotopes. Nuclear extracts and radioactive probes are mixed in a tube. The mixture is then separated on a non-denatured polyacrylamide electrophoresis gel, and the slower migrating bands corresponding to the DNA/protein complexes are visualized relative to the DNA probe. The probes may be double-stranded DNA or single-stranded DNA, which bind to proteins of interest. Purified proteins, partially-purified proteins or nuclear extracts can be used in detecting DNA-binding factors or RNA-binding factors.

To define specificity of the protein/DNA interaction, unlabeled competitive dsDNA probes are added to protein/DNA reaction. These competitive probes compete with the labeled dsDNA probes for binding to the proteins, thus, the specificity of the protein/DNA interaction can be determined with the experiment.

Although the theory of EMSA is very easy, it is necessary to do multiple experiments to attain the optimal reaction system of successful EMSA that is influenced by the origin of protein and binding sites. The follow are the factors that must be optimized: preparation of nuclear extracts (proteinases in the extracts may destroy DNA-binding proteins), concentration of binding proteins, concentration of the probes, concentration of non-specific probes, contents and pH of buffers, condition, temperature and time of incubation, the vectors, etc. The total volume should not be more than 20μl. (dI:dC)(dI:dC).

At present, most EMSA experiments used DNA-probe labeled with the [³²P]-radioisotope and therefore highly radioactive. In addition, the lifetimes of these probes are limited due to the self-destroying radiation and the short half-life of ³²P. By avoiding the radiation-related problem, non-radioactive EMSA kits have been developed. However, the kits using the probes labeled with digoxigenin-ddUTP are less than ideal because of the low sensitivity and weak signal. Other non-radioactive EMSA kits on the market only provide detection reagent, and the specifically DNA or RNA probes must be prepared by end-users. Also, optimal reaction condition must be determined by the end-users by trial and error. Often, a set of EMSA kits will have already been exhausted before any reliable experimental results can be obtained.

With Viagene's non-radioactive EMSA kits, the condition of protein/DNA interaction has been optimized for a specific transcription factors through many experiments. And all these kits are ready to use and guarantee to work.

Viagene’s non-radioactive EMSA kits are based on high sensitivity of the chemiluminescence and biotin labeled technology, sensitivity of which is substantially higher than radioactive EMSA. The end-users may manipulate chemiluminescence-imager systems to do quantitative analyses to the results. Viagene’s EMSA kits contain binding reaction reagents, detection reagents and substrate solutions. Quality is guaranteed for 12 months.

References：

1. Briggs M R et al 1986 Science 234, 47.
2. Lee W et al 1986 Cell 49, 741.
3. Williams T et al 1989 Genes Dev 2, 1557.
4. Sen R and Baltimore D 1986 Cell 46, 705.
5. Parlsow T G et al 1984 Proc Natl Acad Sci USA 81, 2650.
6. Montminy M R et al 1986 Proc Natl Acad Sci USA 83, 6682.

NF-кB was determined to have DNA binding activity with the enhancer of к-chain of immunoglobulin in B cells and was discovered in the cytoplasm of other cell types, which forms compounds of NF-кB/I-кB. The classical NF-кB is a heterodimer composed of a 50-kd (p50) and a 65-kd (p65 or RelA) subunits. Other members of the mammalian Rel family include p75 (c-Rel), p49 (p52) and p78 (RelB).

The subunits of p65. p68 and p75 have high transactivation and p50, p49 have relative low transactivation activity, although they all can bind to DNA.

Some studies have suggested that p49 and p65 may also form a heterodimer with transcriptional activity. The heterodimer of p49 and p65 is similar to that of p50 and p65. An inhibitor IKBα/MAD-3 in the cytoplasm controls the heterodimer of p49/p65 and p50/p65. The compound of IкB/p65 inhibits the transcriptional activity of NFкB in nucleus. In vitro, high concentrations of p65 can form homodimers that can bind DNA slightly, which can be inhibited by Poly(dI:dC). P49 and p50 also could form small quantities of their homodimers in cells.                        

Usually in NF-кB EMSA experiment, the 20ul reaction system contains 10 mM HEPES, 0.28pM NF-кB oligonucleotides, 50mM KCl, 0.2mM EDTA, 2.5mM DTT, 10% Glycerol, 0.05% NP-40. 250-300ng nuclear proteins and 10μg HeLa nuclear extracts are enough to form complexes that must be kept at RT for 30 minutes and separated in 7%polyacrylamide that contains 50mM Tris (pH8.3) and 38mM Glycerol. The loading buffer that contains Coomassie Brilliant Blue G250 and Xylene Cyanol FF must be add to the negative control system. Coomassie Brilliant Blue G250 and Xylene Cyanol FF can break down the structure of complexes of NF-кB in reaction. It can form two kinds of sequence-specific compounds that are homodimers of p50/p50 and heterodimers of p50/ p65 when HeLa nuclear extracts are used instead of purified nuclear proteins. Four distinct complexes (p49/ p49，p50/ p50，p50/ p65，p49/ p65) can be detected in the cells that  are expressing p49, p50 and p65. If the concentration of p65 is high in cells, you may detect some p65/p65 homodimers. The reagent GTP，ATP，Agmatin, Spermidine, Ba²⁺, Ca²⁺, Co³⁺(NH3)6 could enhance the capability of binding of NF-кB.

1. Urban M B et al 1991 EMBO J 10(7), 1817
2. Baeuerle P A 1991 Biochim Biophys Acta 1071, 6                                         

Dominant-negative STAT3 [Cat #：STRG01]
A vector carries a mutant STAT3 gene, which inhibits the transcriptional activation functions of STAT3.

Dominant-negative STAT3/AD [Cat #：STRG01/AD]
An adenoviral vector carries a mutant STAT3 gene, which inhibits the transcriptional activation functions of the STAT3 with high transfection efficiency for most human and animal cells.

Dominant-negative STAT5 [Cat #：STRG02]                                                            
A vector carries a mutant STAT5 gene, which inhibits the transcriptional activation functions of the STAT5.

Dominant-negative STAT5/AD [Cat #：STRG02/AD]
An adenoviral vector carries a mutant STAT3 gene, which inhibits the transcriptional activation functions of the STAT3 with high transfection efficiency for most human and animal cells.

Dominant-negative IκBα/AD [Cat #：STRG03/AD]
An adenoviral vector carries a mutant IkBa gene, which negatively regulates the NFкB function with high transfection efficiency for most human and animal cells.

STAT3/luc [Cat #：SIRP01]
A vector carries a luciferase reporter gene with a promoter, to which STAT3 can bind and induce transcription.

STAT3/luc/AD [Cat #：SIRP01/AD]
An adenoviral vector carries a luciferase reporter gene with a promoter, to which STAT3 can bind and induce transcription with high transfection efficiency.

GAS/luc [Cat #：SIRP02]                                                                                        
A vector carries a luciferase reporter gene with a promoter, to which STAT5 can bind and induce transcription.

GAS/luc/AD [Cat #：SIRP02/AD]
An adenoviral vector carries a luciferase reporter gene with a promoter, to which STAT5 can bind and induce transcription with high transfection efficiency.

PSRE/luc [Cat #：SIRP03]
A vector carries a luciferase reporter gene with a promoter, to which IFN can bind and induce transcription.

PSRE/luc/AD [Cat #：SIRP03/AD]
A vector carries a luciferase reporter gene with a promoter, which IFN can bind to and start transcription.

NFKB/SEPD [Cat #：NFAKP01]
A vector carries a secreted alkline phosphatase reporter gene with a promoter, to which NF-кB can bind and indcue transcription.

NFкB/SEPD/AD [Cat #：NFAKP01/AD]
An adenoviral vector carries a secreted alkline phosphatase reporter gene with a promoter, to which NF-кB can bind and induce transcription.

STAT5/SEPD/AD [Cat #：T5AKP01/AD]
A vector carries a secreted alkline phosphatase reporter gene with a STAT5 promoter.