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SERVICES >  Signaling Pathways > EMSA>>

A. NON-RADIOACTIVE EMSA (Electrophoretic Mobility Shift Assay) Services
[TFASY100, TFASY101]

 

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 of polyacrylamide gel shift, temperature and time of incubation, the vectors, etc. The total volume should not be more than 20μl. Usually, binding buffers contain 4% Glycerol, 1mM MgCl2, 0.5mM EDTA, 0.5mM DTT, 50mM NaCl, 10mM Tris-HCl (pH7.5), 0.05mg/ml poly (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.
 

　
◊ Non-Radioactive NF-кB Services [Cat#: TFASY100] 

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 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 sightly, 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 10ug 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 complexer 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.

References：  
  1. Urban M B et al 1991 EMBO J 10(7), 1817
  2. Baeuerle P A 1991 Biochim Biophys Acta 1071, 63
　
◊ Non-radioactive STAT5 Service [TFASY100]

 

STAT (Signal Transducer and Activator of Transcription) are intracellular molecules that normally are phosphorylated and activated as a result of growth factors inducing receptor dimerization, followed by phosphorylation of receptor-bound JAKs (janus kinases), which in turn phosphorylate the intracellular portion of growth factor receptors, to which inactive STAT molecules bind and themselves become phosphorylated. Once STAT molecules are phosphorylated, they rapidly form dimers or tetramers, which permits them to be transported into the nucleus, where they bind to specific nucleotide sequences in the regulatory regions of endogenous genes and activate or inhibit transcription of those genes. For example, STAT5A and STAT5B bind to the sequence TTNNNNNAA, which most often is TTCNNNGAA, with "N" meaning any nucleotide. STAT3 binds preferentially to a similar sequence with one fewer "N" - TTNNNNAA. STATs 5A and 5B are the products of two separate (presumably duplicated) genes adjacent to each other on human chromosome 17, which are 95% homologous. Their expression and activation in cells may or may not be closely coordinated. Although STATs 5A and 5B target mostly the same genes, there are some differences, which presumably are based on the small differences between STAT5A and 5B and different nucleotides within the internal "NNN" and flanking  ) the target nucleotide sequences. There also are many genes that are regulated by both STAT5 and STAT3.

 

It has been discovered that many malignancies are associated with the perhaps driven by aberrant, constitutive activation of STAT5 (e.g., acute myeloid leukemias, acute lymphoblastic leukemias, chronic myelogenous leukemias pbcr/abl activated STAT5], certain head and neck carcinomas) and STAT3 (e.g., multiple myeloma, breast cancers).

 

Viagene's non-radioactive STAt5 EMSA services detect activated STAT5 dimers. Although it may be possible under optimal conditions to distinguish STAT5A and STAT5B from each other by the slight differences in their molecular weight and electrophoretic migration in polyacrylamide gels, this can be difficult and is notrecommended as the sole means to distinguish between these two molecules. For definitive identification of activated STAT5A and STAT5B, EMSA supershift assays should be carried out using antibodies that react specifically with either STAT5A or STAT5B.

 References：

  1. Imada K,Leonard W J.The JAK-STAT pathway.Mol Immunol, 2000, 37:1-11
  2. Magrassi L,De Fraja C,Conti L,et al.Expression of the JAK and STAT superfamilies in human meningiomas. J Neurosurg, 1999,91:440-446
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◊ EMSA service for the probes Viagene don't have, please contact us.

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Phone 813-902-3827, 813-679-7555: Fax 813-830-7389 
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