Electrophoretic mobility shift assay history

Principle[ edit ] A mobility shift assay is electrophoretic separation of a protein—DNA or protein—RNA mixture on a polyacrylamide or agarose gel for a short period about 1. Under the correct experimental conditions, the interaction between the DNA or RNA and protein is stabilized and the ratio of bound to unbound nucleic acid on the gel reflects the fraction of free and bound probe molecules as the binding reaction enters the gel. This stability is in part due to a "caging effect", in that the protein, surrounded by the gel matrix, is unable to diffuse away from the probe before they recombine. If the protein concentration is not known but the complex stoichiometry is, the protein concentration can be determined by increasing the concentration of DNA probe until further increments do not increase the fraction of protein bound.

Electrophoretic mobility shift assay history

They found that CTNS encodes an integral membrane protein, which they designated cystinosin, that has features of a lysosomal membrane protein. CTNS, a putative cystine transporter, contains amino acids and 7 transmembrane domains.

Electrophoretic mobility shift assay history

This region contains an Sp-1 regulatory element at positions to which, as shown by electrophoretic mobility shift assays, binds Sp The most common mutation was a kb deletion Of 82 alleles bearing the kb deletion, 38 derived from Germany, 28 from the British Isles, and 4 from Iceland.

The 18 new mutations identified in this study included the first reported missense mutation, 2 in-frame deletions, and mutations in patients of African American, Mexican, and Indian ancestry.

CTNS mutations were spread throughout the leader sequence, transmembrane, and nontransmembrane regions. According to a cystinosis clinical severity score, homozygotes for the kb deletion and for WX had average disease, whereas mutations involving the first amino acids prior to transmembrane domains were associated with mild disease.

Electrophoretic mobility shift assay history

By Northern blot analysis, CTNS was not expressed in patients homozygous for the kb deletion but was expressed in all 15 other patients tested. Structure predictions suggested that cystinosin is a novel integral lysosomal membrane protein.

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They screened patients with infantile nephropathic cystinosis, those with late-onset cystinosis, and patients whose phenotype did not fit the classic definitions. They identified 23 different mutations in the CTNS gene, 14 of which were novel. Of 25 patients with infantile nephropathic cystinosis, 12 had 2 severely truncating mutations, consistent with a loss of functional protein, and 13 had missense or in-frame deletions, which would result in disruption of transmembrane domains and loss of protein function.

Mutations identified in 2 late-onset patients see, e.

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For 3 patients, the age of onset of cystinosis was under 7 years, but the course of the disease was milder than the infantile nephropathic form. This suggested that the missense mutations identified in these individuals see, e. As indicated earlier, identification of the CTNS gene was facilitated by the detection of deletions spanning the cystinosis locus in affected individuals.

Two types of deletions were detected, one of approximately 65 kb, which was found in homozygous state in nearly one-third of cystinotic individuals, and a smaller one of 9.

Both the larger and the smaller deletion span the 5-prime end of the CTNS gene, covering exons 1 to 10 and 1 to 3, respectively; STS analysis indicated that the larger deletion was the same size in all patients. This type of mechanism suggested that the deletion of approximately 65 kb is not a recurrent mutation, and the results confirmed that it is identical in all patients.

Haplotype analysis showed that this large deletion is due to a founder effect that occurred in a white individual, and that it probably arose in the middle of the first millennium. Each of the promoter mutations drastically reduced CAT activity when inserted into a reporter construct and failed either to cause a mobility shift when exposed to nuclear extract or to compete with the normal oligonucleotide's mobility shift.Poly(N-isopropylacrylamide): experiment, theory and applicationAuthor links open overlay panel H.G.

Schild. Show more. Town et al. () determined that the CTNS gene has 12 exons. Phornphutkul et al.

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() identified the CTNS promoter as the region encompassing nucleotides to +1 with respect to the transcription start site. This region contains an Sp-1 regulatory element at positions to which, as shown by electrophoretic mobility shift assays, binds Sp Development.

This technique was patented in by William J. Littlehales under the title "Electroblotting technique for transferring specimens from a polyacrylamide electrophoresis or like gel onto a membrane.

Town et al. () determined that the CTNS gene has 12 exons. Phornphutkul et al. () identified the CTNS promoter as the region encompassing nucleotides to +1 with respect to the transcription start site. This region contains an Sp-1 regulatory element at positions to which, as shown by electrophoretic mobility shift assays, binds Sp Development.

This technique was patented in by William J. Littlehales under the title "Electroblotting technique for transferring specimens from a polyacrylamide electrophoresis or like gel onto a membrane.

Molecules, an international, peer-reviewed Open Access journal.

Electroblotting - Wikipedia