Method and composition for quantifying protein using tagged standards

Patent metadata

Field	Value
Publication number	US-12259356-B2
Application number	US-202117544591-A
Country	US
Kind code	B2
Filing date	Dec 7, 2021
Priority date	Dec 7, 2020
Publication date	Mar 25, 2025
Grant date	Mar 25, 2025

Abstract

Methods and reference compositions for quantifying protein using tagged standards. In an exemplary method, a reference composition and a protein may be electrophoresed in respective lanes of a gel. The reference composition may include quantitation standards of different size and each including a tag present at a different concentration. The quantitation standards and the protein may be transferred from the gel to a solid support to create a blot. Luminescence may be detected from the blot to obtain respective luminescence values separately representing an abundance of the tag of each quantitation standard and an abundance of the protein. A quantity of the protein may be determined using the respective luminescence values.

First claim

We claim:

1. A method of quantifying a protein, the method comprising:
electrophoresis reference composition and the protein in respective lanes of a gel, the reference composition including quantitation standards of different size each including a tag present at a different concentration;
transferring the quantitation standards and the protein from the gel to a solid support to create a blot;
detecting luminescence from the blot to obtain respective luminescence values separately representing an abundance of the tag of each quantitation standard and an abundance of the protein, wherein detecting luminescence is performed using plural luminescence images representing luminescence detected from the blot for different amounts of time; and
determining a quantity of the protein using the respective luminescence values, wherein determining a quantity uses a first luminescence value obtained from a first luminescence image representing a shorter detection time and a second luminescence value obtained from a second luminescence image representing a longer detection time, and wherein the first luminescence value corresponds to a first quantitation standard having a higher concentration of the tag and the second luminescence value corresponds to a second quantitation standard having a lower concentration of the tag.

2. The method of claim 1 , further comprising determining whether the luminescence value for the protein falls within a linear range of luminescence response based on the luminescence values for the quantitation standards.

3. The method of claim 1 , wherein detecting luminescence is performed using a nondestructive read sensor.

4. The method of claim 1 , wherein determining a quantity of the protein includes producing a standard curve using the luminescence values for the quantitation standards, identifying a point along a linear portion of the standard curve, the point having a luminescence value matching the luminescence value of the protein, and finding a quantity value associated with the point.

5. The method of claim 1 , wherein the quantitation standards are calibrated against a known amount of the protein, such that the luminescence value for the tag of each quantitation standard corresponds to a defined amount of the protein.

6. The method of claim 1 , wherein determining a quantity of the protein includes producing a standard curve using the luminescence values for the quantitation standards, wherein detecting luminescence is performed using an image sensor having a dynamic range, and wherein the standard curve encompasses a range of tag concentrations that is greater than the dynamic range of the image sensor.

7. The method of claim 1 , wherein detecting luminescence includes detecting chemiluminescence representing each of the quantitation standards and the protein.

8. The method of claim 1 , wherein detecting luminescence includes detecting fluorescence representing each of the quantitation standards and the protein.

9. The method of claim 1 , wherein at least two of the tags of the quantitation standards differ in concentration by at least 100-fold.

10. The method of claim 9 , wherein the at least two tags of the quantitation standards differ in concentration by at least 1000-fold.

11. The method of claim 1 , wherein the reference composition also includes a set of size markers each including a label that is chromophoric or fluorescent, and wherein each quantitation standard corresponds in size to one of the size markers.

12. The method of claim 11 , wherein the label of each size marker is a chromophoric label, further comprising capturing an image representing absorbance of light by the chromophoric label of each size marker.

13. The method of claim 1 , wherein the tags of the quantitation standards are structurally identical to one another.

Assignees

• Bio Rad Laboratories Inc

Inventors

• Oh Kenneth
• Marlowe Carl
• Swihart Stephen L
• Thrush Evan
• Griffin Michael

Classifications

• G01N33/6827

  Total protein determination, e.g. albumin in urine · CPC title

• G01N21/6456

  Spatial resolved fluorescence measurements; Imaging · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• G01N33/582

  with fluorescent label · CPC title