EMSA is a very popular technique to detect activated transcriptional factors (TF) and DNA- or RNA-binding proteins. With the “standard” radioactive EMSA techniques using P32-labeled oligonucleotides, x-ray film and film developers, the results can only be obtained after laborious procedures and 2-3 days of film exposure time, working with radioactive materials. Even with non-radioactive EMSA with DIG-labeled oligonucleotides, the experimental result still can be obtained only after 3 days.
With Viagene's non-radioactive EMSA kits (standard electro-transfer), the detection can be shorten to ~7 hours. With years of intensive development work by Viagene Biotech scientists, EMSA assays based on biotin-labeled probes can be reduced to ~5 hours (semi-dry transfer). With our most advanced EMSA assay based on infra-red (IR) fluorescence (IR-EMSA) developed by our scientists, an EMSA assay can be completed in ~2 hours. The IR-EMSA is not only a rapid way to perform EMSA, but the operation also is much easier than that of other detection methods (comparison of different EMSA assays), becoming the easiest and fastest way to detect the activation of transcription factors and other DNA/RNA-binding proteins.
A. The principle of the Viagene 2-hour EMSA (Rapid EMSA):
Rapid EMSA is based on the use of probes labeled with infrared (IR) fluorescence. The principle is easy to understand: IR-labeled probes are much smaller chemicals than that of probe/TF complexes and move fast in non-denatured polyacrylamide gels, whereas the much larger probe/TF complexes would migrate more slowly and would localize at a higher position in the gel. The location of fluorescent-probes can be detected by imagers or scanners (Pic. on the right)
B. Key features of the Viagene 2-hour EMSA:
2) Easy operation: no transfer to membranes, no crosslinking, no blocking & no need for HRP and substrates.
3) Less aggregation
4) Fast results.
5) Capable of quantitation.
In spite of the advanced features and advantages of the 2-hour IR labeling-based EMSA described above, it may not suitable for everyone, because of the following limitation and considerations:
C. The problem with 2-hour EMSA:
1）Need for a specific detection device – Non-radioactive fluorescent EMSA has been performed in limited numbers of laboratories for quite some time, but it was limited by the problem of low sensitivity until the IR-imager was introduced by Li-Cor. Viagene's 2-hour rapid EMSA kits require the use of Li-Cor's IR-imager: Odyssey 9120 or Odyssey Cx.
2）IR Fluo-probes are more expensive: Compared to the preparation of Biotin-labeled EMSA probes, only very few companies provide fluorescence-labeling service for oligonucleotides, which cause the IR-dyes to be more expensive. If ordered from the major supplier, the other company charges $90-280 for each end-labeling plus the cost for DNA synthesis. For ordering an double-stranded DNA probe labeled with IR Fluorescent dyes for EMSA would cost at least $265, which is only for two single-strand DNA synthesis and labeling, but not includes double-stranded aligning, labeling efficiency testing and confirmation, and determination of optimal procedural conditions.
3）Probe-related issues: Due to their chemical structure, IRFluo including IR-Dye 700, IR-Dye 800 and CY5.5..., tend to form aggregates in the loading wells and thus have a weak binding signal for IR-Fluoro EMSA (See a photo of an example of such aggregates in a 4-12% gel used for NFkB EMSA on the Li-Cor website). Many research labs gave up trying to use the IR-Fluoro EMSA because of this problem.
D. Vingene's solution for IR-EMSA:
At Viagene, our custom IR-fluorescence labeling oligonucleotide probe service includes probe design, DNA synthesis, IR-Fluoro labeling, HPLC purification, double-stranded DNA alignment, confirmation of double-stranded DNA formation, confirmation of labeling efficiency, and determination of the optimal working-dilutions. With Viagene's IR-Fluoro EMSA kits, there is a marked reduction in aggregate formation in the EMSA gels (compare the above EMSA photo from Viagene Biotech with those from other companies). For all of these services to provide superior IR-fluorescence-labeled, double stranded oligonucleotide probes, Viagene charges less than that of other competitor companies which provide products without all of these quality checks and without optimization or clear guidance on optimal experimental conditions to aid researchers in performing their EMSA experiments successfully. The custom IR-fluorescence probe service provided by Viagene requires ~7 working days for preparation, and our customers will get the probes in working condition (ready to use) with clear procedural instructions.
E. Requirements for 2-hour rapid IR-Fluoro-based EMSA:
Purified samples or cell extracts with
activated DNA- or RNA-binding proteins (Viagene's
nucleus extraction kit).
2) IR-Fluoro-labeled oligonucleotide probes for EMSA, which are included with complete EMSA kits (see the list of kits or ordered via Custom-synthesis.
3) IR-Fluoro EMSA reagent kit (Viagene's IRFluo EMSA kits).
4) Reagents and equipment for vertical gel electrophoresis (see Viagene's reagents, devices, and ready gels).
5) ) Li-Cor Odyssey IR-Fluorescence Scanner 9120/CX or any device with capacity for scanning infrared dyes at 670-690nM.
F. Related products.
|General EMSA kit for using IR Fluo-DNA probes.||SIDET201||100||299|
|General EMSA kit for using IR Fluo-RNA probes.||SIDET202||100||356|
|IR EMSA kit||Compete EMSA kits with an IR Fluo-DNA probe.||IRTFxxx
|IR Fluo-probes||Ready IR Fluo-labeled probes.||TFxxxIR
|Custom IR Fluo-DNA probes||IRSRV01
|Custom IR Fluo-RNA probes||IRSRV03