Labeling proteins with dyes that are insoluble or only sparingly soluble in water

a technology of protein labeling and dye, applied in the direction of instruments, enzymology, material analysis through optical means, etc., can solve the problems of uniformity among the various different proteins, inability to remove excess dye, labor-intensive and time-consuming procedure, etc., and achieve the effect of minimizing the separation behavior of each protein

Inactive Publication Date: 2005-12-29
BIO RAD LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0005] It has now been discovered that fluorescent dyes that are insoluble or at most sparingly soluble in water can be applied to biological samples in an unusually efficient manner by using the dye in solid dry form in the presence of a non-nucleophilic buffering agent and in the absence of organic solvents. In certain embodiments of the invention, a denaturing agent is also present. The solid dry composition can be either a powder that is added to the biological sample or a dry coating on the inner surface of a sample tube or other receptacle, such as a test tube or cuvette, in which the sample is placed. When the composition is a powder, it preferably contains the buffering agent and the denaturing agent and is suspended in the liquid sample. When the composition is a coating on the inner surface of a receptacle, the coating preferably contains the dye plus a polymer that controls the rate of dissolution of the dye into the sample, and t

Problems solved by technology

Applying the dyes in this manner and removing excess dye is a labor-intensive and time-consuming procedure that is susceptible to handling difficulties, operator error and various nonuniformities.
One difficulty with these methods is that the ratio of dye to protein must be carefully controlled to avoid overstaining the proteins.
Overstaining can cause nonuniformities among the various different proteins in the amount of dye that is coupled to each protein.

Method used

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Embodiment Construction

[0008] Dyes that can be used in the solid dry composition of this invention include fluorescent dyes whose solubility in water is less than 5×10−6 g / cc (i.e., less than 5 μg / mL), preferably less than 0.3×10−6 g / cc (i.e., less than 0.3 μg / mL). Preferred dyes are those that can be excited by light at a wavelength between 400 nm and 700 nm, since glass and plastic, the materials used in the construction of electrophoresis gel cassettes, absorb and fluoresce less within this wavelength range.

[0009] Included among these dyes are electrophilic-activated forms of fluorescent dyes including, but not limited to, succinimidyl esters, vinyl sulfones, etc., of xanthenes, cyanines, coumarins, benzimides, phenanthridines, ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, and quinoline dyes. Succinimidyl groups are of particular interest as the reactive group on the dye, since the succinimidyl group reacts efficiently with a primary amino group on a peptide, and succ...

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Abstract

The proteins in a biological sample that is sought to be analyzed for its protein composition by an electrophoretic or chromatographic procedure are coupled to a dye in an unusually efficient manner by combining the sample with a solid dry composition containing the dye a buffering agent, and in preferred embodiments, a denaturing agent as well. The solid and dry form of the composition prevents the dye from deteriorating or decomposing, and the combination of components in the composition allows the dye to couple to the proteins in a relatively uniform manner with no overstaining of the protein when the composition and the sample are heated together and held at an elevated temperature for a short period of time.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation-in-part of co-pending application Ser. No. 09 / 645,784, filed Aug. 24, 2000, which application is incorporated herein in its entirety for all purposes capable of being served thereby.BACKGROUND OF THE INVENTION [0002] The analysis of biological samples to determine the identity and amounts of various proteins for diagnostic or research purposes is performed by a wide variety of separation techniques, many of which involve the use of dyes to enable the clinician to locate, identify, and quantitate the proteins. The dyes are detectable and readable by visual observation or are machine-readable. Included among the many types of dyes that are used for this purposes are those that emit colors in the visible range or the ultraviolet range to the eye without treatment or activation, and fluorescent dyes which emit upon excitation. [0003] The dyes are most commonly applied to the proteins after separation has oc...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N21/76G01N21/78G01N27/447G01N33/483G01N33/543G01N33/68
CPCG01N33/54366G01N33/6839Y10T436/13Y10S435/968Y10S436/80G01N2021/6439
InventorZHU, MINGDEOLECH, LEE
OwnerBIO RAD LAB INC