After the shift bands of protein-nuclear acid complexes are observed in EMSA, Two common approaches, Competitive EMSA or/and Supershift EMSA (detail in other section), are used to determine the specificity of protein and nuclear acid interaction.
1. What is the Competitive EMSA? Why need it?
Competitive EMSA is the most common test of specificity. Prior to the addition of p32-, biotin- or fluorescent dye-probes, a 50-100 fold molar excess of unlabeled competitor DNA is added to the reaction mix. Individual reactions are performed with oligonucleotides containing the target DNA sequence and oligonucleotides which have been specifically mutated within the target sequence. Specific binding is indicated by a loss of factor binding to the biotin- or fluorescent dye-probes. The alteration of conserved bases within the binding site can abolish the ability of a transcription factor to bind to its cognate DNA. Thus for site-directed mutant competitions, binding of the labeled probe is preserved. In addition, competitions should also be performed with well characterized consensus DNA sequences specific for the factor of interest.
2. How to design specific competitive or mutant probes?
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.
Designing a mutant probe follows these rules: 1) muting one or more bases in the binding domain of consensus sequences. 2) Usually, changing AT to GC, or vice versa. 3) checking mutant probes to avoid forming another binding domain by making adjustment. 4) Verifying mutant-probes with EMSA.
3. How to perform Competitive EMSA?
In order to get an expecting result from Competitive EMSA, the experiments should consider following points:
(1) The cell or tissue
extracts should have activated proteins/factors enabled to
bind to DNA or RNA probes.
(2) Shifted bands of protein-nuclear acid complexes must be detected in conventional EMSA.
(3) Competitive or mutant probes should be added to reaction mixture before the reaction of proteins and DNA/RNA.
(4) Nuclear proteins used for EMSA are isolated with a high-salt buffer. High volume of nuclear extracts may interrupt protein/nucleic acid interaction.
(6) The radio of competitive probes/labeled probes varies significantly among different DNA/RNA binding proteins. The ratio of 1:20 may cause serious competition for one protein factor, but as high up to ratio of 1:200 may require to producing visible competition for another protein factor.
(7) A classic Competitive EMSA may consider to include these reactions: 1) A sample without activated target proteins (negative sample) + labeled probes, 2) A sample with activated target proteins + labeled probe (positive sample), 3) positive sample + labeled probe + low dose of specific competitors (competitive low), 4) positive sample + labeled probe + high dose of specific competitors (competitive high), 5) positive sample + labeled probe + low dose of mutant probes (mutant low), 6) positive sample + labeled probe + low dose of specific competitors (mutant high).
4. Interpretation of Competitive EMSA.
The result of Competitive EMSA can be judged by; 1) the positive competition for specific probe should be that shifted bands become weak or disappearing, but non-specific bands are not affected, when specific competitive probes are applied. 2) The specificity of protein-nucleic acid interaction is further confirmed by mutant competitive probes; both specific shifted bands and non-specific bands should not be affected when mutant competitive probes are used. Picture 2 shows a sample of Competitive EMSA (Blood 1999, 93:2369-79).
5. Common problems with Competitive EMSA
(1) No competition with unlabeled probes, which could be caused by
1) the complexes are formed by
non-specific interaction. 2) no enough competitive probes are used.
The radio of competitors/labeled probes varies among
different binding proteins; the ratio of 1:20 may
cause serious competition for one protein factor, but as
high up to the ratio of 1:200 may require to producing visible
competition for other protein factors. 3) amount of labeled
probes is used too much.
(2) Both target and non-specific bands disappear when unlabeled probes are used, which could be caused by too much competitive probes being used.
(3) Mutant probes cause significant competition, which could be caused by 1) the point-mutation of binding domain is not good enough to prevent the binding of the probe to protein factors. 2) the amount of mutant probes is used too much.
In any case, if non-specific bands become weak or disappearing, indicating amounts of competitive or mutant probes are used too much.
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