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Methods, devices and systems for characterizing proteins

A technology of microfluidic devices and channels, which is applied in the field of characterizing proteins, devices and systems, and can solve problems such as capillary system labeling and separation difficulties

Inactive Publication Date: 2007-02-07
CAPLIPER LIFE SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Also, the presence of SDS in protein separations (which guarantees size-based separations) makes labeling and separation more difficult in capillary systems

Method used

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  • Methods, devices and systems for characterizing proteins
  • Methods, devices and systems for characterizing proteins
  • Methods, devices and systems for characterizing proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Embodiment 1: Separation of polypeptides with SubCMC separation buffer

[0111] Fluorescence data received from the separation channel were recorded by a computer (PC equipped with an Intel Pentium(R) microprocessor). Data are displayed as a linear plot of fluorescence versus time and in the form of a simulated gel generated with Caliper Technologies Corp. proprietary software.

[0112] A 0.5M Tris-Tracen buffer solution was prepared by dissolving Trisine in deionized water to a concentration of 0.5M, then adjusting the pH to 7.5 with 1M Tris. The resulting buffer was then filtered through a 0.22 μm syringe filter. Prepare sieving or separation buffer with 3% polydimethylacrylamide-co-acrylic acid dissolved in 12.5 mM Tris-Traxine buffer containing 0.9% (w / v) sodium dodecyl sulfate (SDS) , and 10 μM Syto 60 dye (Molecular Probes, Eugene OR). Then, through Costar Spin-X TM Separation buffer was filtered through a 0.22 μm cellulose acetate centrifugal filter.

[011...

Embodiment 2

[0125] Example 2: Isolation and Detection of Peptides Using Post-Isolation / Pre-Assay Dilution

[0126] Such as Figure 7 The microfluidic device shown is filled with separation buffer, as described above. Separation channel 704 intersects diluent channels 720a and 722a at a point 1.2 cm downstream of injection point and 0.1 cm upstream of detection point 732 . Separation buffer contained 4.2% non-crosslinked polydimethylacrylamide / co-acrylic acid in 30 mM Tris trisine buffer and 0.13% SDS. Dilution buffer containing 30 mM Tris-Traxine but no polymer or SDS was added to reservoirs 720 and 722 . The buffer is flowed into the separation channel by an electrokinetic method, such as electrophoresis.

[0127] Polypeptide standard solutions (10-205 kD protein standards from Bio-Rad, Inc.) are added to a sample reservoir, such as reservoir 706, loaded and injected into the separation channel using the same method described in US Pat. No. 5,976,336 (previously incorporated herein). ...

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Abstract

A method of characterizing a polypeptide, comprising providing a first capillary channel having a separation buffer disposed within, wherein the separation buffer comprises a non-crosslinked polymer solutin, a buffering aten, a detergent, and a lipophilic dye. The separation buffer is provided such that, at the time of detection, the detergent concdntration in the buffer is not above the critical micelle concentration. The polypeptide is introduced into one end of the capillary channel. An electric field is applied across a length of the capillary channel, which transports polypeptides of different sizes through the polymer solution at different rates. The polypeptides is then detected as it passes a point along the length of the capillary channel.

Description

Background of the invention [0001] Any effort to understand life, the processes that sustain it, and the events and factors that influence these processes must characterize biological compounds. In general, understanding living processes and achieving their control focuses first on the fundamental building blocks of life, the macromolecular compounds and complexes that distinguish living organisms from lifeless native oozes. Of particular interest in understanding and controlling living processes are nucleic acids and the proteins they encode. [0002] In the case of proteins, many characterization methods have remained largely unchanged over the past few decades. For example, current protein characterization methods generally rely, at least in part, on sodium dodecyl sulfate polyacrylamide gel electrophoresis or SDS-PAGE to characterize proteins by relative molecular weight. These methods employ a plate or sheet of cross-linked polyacrylamide. Proteins to be separated and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26C07K1/24G01N27/447
CPCC07K1/24G01N27/44791G01N27/44747
Inventor A·乔B·法索拉赫J·C·小米克尔森M·A·斯佩蒂A·维诺托
Owner CAPLIPER LIFE SCI INC