Protein expression profiling

A purpose and analyte technology, applied in the direction of analytical materials, microbial measurement/inspection, instruments, etc., can solve problems such as difficult to simplify operations, small size, etc.

Inactive Publication Date: 2003-10-01
MOLECULAR STAGING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is difficult to simplify, and due to the rather small sample volume (about 0.5-5nl) required to generate spots in these microarrays, very sensitive analytical methods must be used

Method used

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  • Protein expression profiling
  • Protein expression profiling
  • Protein expression profiling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] This example illustrates the construction and characterization of antibody DNA conjugates used as Reporter Binding Primers

[0182] Oligonucleotides. All oligonucleotides used were synthesized by Perseptive Biosystems Rapid DNS Synthesizer and purified by reverse phase HPLC. Circular DNA was constructed as previously described (4). Conjugate rolling circle replication primer: 5'-thiol-GTA CCA TCA TAT ATG TCC GTG CTA GAA GGA AACAGT TAC A-3'; amplified target circular DNA: 5'-TAG CAC GGA CAT ATA TGA TGG TAC CGCAGT ATG AGT ATC TCC TAT CAC TAC TAA GTG GAA GAA ATG TAA CTG TTTCCT TC-3'; Detection Probe-5'Cy3 TAT ATG ATG GTA CCG CAG Cy3 3', 5'Cy3TGA GTA TCT CCT ATG ACT Cy33', 5'Cy3 TAA GTG GAA GAA ATG TAA Cy33.

[0183] Antibody-DNA Conjugates. Antibodies were buffer exchanged into a buffer of 50 mM sodium phosphate pH 7.5, 150 mM NaCl, 1 mM EDTA by chromatography on a PD-10 column (Amersham-Pharmacia Biotech). Desalted antibody (41 nmoles) was treated with a 10-fold mola...

Embodiment 2

[0187] This example illustrates the use of indicator antibody primers / antibody-DNA conjugates for the detection of analytes in an ELISA assay. Compared with the use of conventional antibody-enzyme conjugates, the immuno-RCA assay showed high sensitivity and good kinetic range.

[0188] ELISA test. The 96-well plate was covered with goat polyclonal anti-human IgE at 100 μl 2 mg / ml per well for 2 hours at 37°C, then overnight at 4°C. The plate was washed 3 times with 100 μl TBS / 0.05% Tween20, and then blocked with 5% non-fat dry milk for 2 hours at 37°C. Plates were washed again with TBS / 0.05% Tween20 before addition of IgE analyte at various concentrations in a volume of 100 μl. After incubation at 37° C. for 30 min, the plate was washed 3 times with 100 μl TBS / 0.05% Tween20. In a conventional ELISA assay, anti-human IgE-alkaline phosphatase conjugate was added to each well and incubated at 37°C for 30min. After washing the plate with TBS / 0.05% Tween20, the alkaline phospha...

Embodiment 3

[0190] This example illustrates the use of indicator antibody primers / antibody-DNA conjugates to detect analytes in microparticle form. Human IgE was still chosen as the test analyte, but this sandwich assay was performed using avidin-coated magnetized microparticles and a biotinylated polyclonal anti-human IgE capture antibody. In general, streptavidin-coated magnetized beads (Bangs Laboratories) were covered with a solution of 16 μg / ml biotinylated polyclonal anti-human IgE (Pharmingen) in TBS and washed with TBS / 0.05% Tween20 for 3 Once, block overnight with 2mg / ml BSA. The beads were incubated with human IgE (25 ng / ml) for 20 min at room temperature in TBS and washed 3 times with TBS / 0.05% Tween20. IgE detection using conventional anti-IgE-DNA conjugates or immuno-RCA conjugates was performed as described above for the ELISA assay. In Figure 7, it can be seen that in the presence of moderate concentrations of IgE coupled to microparticles, immuno-RCA with anti-IgE-DNA co...

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Abstract

Compositions and methods for detecting small quantities of analytes such as proteins and polypeptides are disclosed. The method involves linking a primer to an analyte, which is then used to mediate rolling circle replication of circular DNA molecules. The replication of DNA circles is dependent on the presence of primers. Thus, the methods disclosed herein can generate an amplified signal from any analyte of interest by rolling circle replication. The amplified DNA remains attached to the analyte via the primer, allowing stereoscopic detection of the analyte. The methods of the invention can be used to detect and analyze proteins or polypeptides. Various proteins can be analyzed by microarrays to which various proteins are immobilized. Thus, rolling circle replication primers are linked to various proteins using a conjugate of the primer and a molecule capable of specifically binding the protein to be tested. Rolling circle replication of the primers produces a large amount of DNA product at the site in the matrix where the protein is anchored. The amplified DNA serves as a signal for easy detection of the protein. The methods of the invention can also be used to compare proteins expressed in two or more samples. The resulting information is similar to the type of information gathered in nucleic acid expression systems. The method of the present invention can detect and quantify the protein expressed in any cell or tissue sensitively and accurately.

Description

technical field [0001] The invention relates to the field of detection and mapping of proteins and polypeptides, especially the field of mapping of trace protein expression. Background technique [0002] The information of the genome is carried by deoxyribonucleic acid (DNA). The size and composition of a given genome sequence determine the form and function of its resulting organism. In general, the complexity of the genome is proportional to the complexity of the organism. Relatively simple organisms such as bacteria have about 1-5 megabases and mammalian genomes are about 3000 megabases. The genome is usually divided into separate regions, known as chromosomes. The bacterium, E. coli, contains a single circular chromosome, while the human genome consists of 24 chromosomes. [0003] Genomic DNA is a double-stranded polymer containing four DNA bases (A, G, C, and T) linked by a sugar-phosphate backbone. The sequence of bases in DNA is the primary sequence structure of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12N15/09C12Q1/68G01N37/00
CPCC12Q1/6804C12Q2563/179C12Q2531/125
Inventor 斯蒂芬·肯斯默吉里什·纳勒巴里·施韦策
Owner MOLECULAR STAGING
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