Multifunctional protein molecular weight ladders

Patent metadata

Field	Value
Publication number	US-10365290-B2
Application number	US-201715491905-A
Country	US
Kind code	B2
Filing date	Apr 19, 2017
Priority date	Sep 19, 2014
Publication date	Jul 30, 2019
Grant date	Jul 30, 2019

Abstract

Multifunctional molecular weight protein ladders and methods of making thereof are disclosed herein that are useful for determining the molecular weight of a test protein and/or the relative mass or amount of the test protein in a protein separation assay, such as gel electrophoresis or western blotting. Also included are compounds of Formula I (e.g., mono acetylated MP-11 NHS ester) that may be used to label purified proteins of the protein ladder. The MP-11 label protein ladder can be detected on a blotting membrane by exposing the microperoxidase to a suitable substrate, such as a chromogenic substrate or a chemiluminescent substrate.

First claim

What is claimed is:

1. A compound having the formula:

wherein n is 0-10 and;
R 1 and R 2 are each independently a member selected from the group consisting of hydrogen and an acetyl (Ac), with the proviso that one of R 1 or R 2 is acetyl and one of R 1 or R 2 is hydrogen.

2. The compound of claim 1 , wherein n is 4, R 1 is hydrogen and R 2 is acetyl.

3. The compound of claim 1 , wherein n is 4, R 1 is acetyl and R 2 is hydrogen.

4. A kit for preparing the protein standard, the kit comprising a plurality of purified proteins of differing molecular weight, a compound, and a labeling buffer, wherein the compound has the formula:

wherein n is 0-10 and;
R 1 and R 2 are each independently a member selected from the group consisting of hydrogen and an acetyl (Ac), with the proviso that one of R 1 or R 2 is acetyl and one of R 1 or R 2 is hydrogen.

5. The kit of claim 4 , wherein the labeling buffer is an amine-free buffer.

6. The kit of claim 4 , wherein the labeling buffer contains SDS.

7. The kit of claim 4 , further comprising a storage buffer.

8. The kit of claim 4 , wherein the storage buffer is substantially free of DTT, EDTA and NaN 3 .

9. A method for preparing a multifunctional protein standard comprising a plurality of purified proteins labeled with at least two chemical moieties selected from the group consisting of a visible dye moiety, a catalytic moiety, a fluorescent dye moiety, a chemiluminescent moiety, and a combination thereof, said method comprising:
(a) determining the optimum number of molar equivalents of the at least two chemical moieties based on the average molecular weight of the plurality of purified proteins when labeled individually;
(b) preparing the plurality of purified proteins in a buffer amenable to the conjugation chemistries of the at least two chemical moieties; and
(c) incubating the at least two chemical moieties and the plurality of purified proteins under conditions such that the at least two chemical moieties covalently attach to the proteins to produce the multifunctional protein standard; wherein one of the at least two chemical moieties is the catalytic moiety, wherein the catalytic moiety is a monoacetylated MP-11 NHS ester having the formula:

Wherein n is 1-10 and;
R 1 and R 2 are each independently a member selected from the group consisting of hydrogen and an acetyl (Ac), with the proviso that one of R 1 or R 2 is acetyl and one of R 1 or R 2 is hydrogen.

10. The method of claim 9 , further comprising purifying the multifunctional protein standard.

11. The method of claim 9 , wherein purifying the multifunctional protein standard comprises size exclusion chromatography or dialysis.

12. The method of claim 9 , wherein incubating the at least two chemical moieties and the plurality of purified proteins comprises simultaneously conjugating the chemical moieties to the proteins.

13. The method of claim 9 , wherein incubating the at least two chemical moieties and the plurality of purified proteins comprises sequentially conjugating the chemical moieties, wherein the monoacetylated MP-11 NHS ester is conjugated to the proteins after the proteins are conjugated to a chemical moiety without having an NHS ester.

14. The method of claim 9 , wherein incubating the at least two chemical moieties and the plurality of purified proteins comprises sequentially conjugating the chemical moieties, wherein the order of conjugating is from the least to most molar equivalents needed for each chemical moiety.

15. The method of claim 9 , wherein at least two purified proteins of the multifunctional protein standard are labeled with the catalytic moiety and the visible dye moiety.

16. A method for performing a Western blot analysis, the method comprising
(a) loading a protein standard comprising a plurality of purified proteins of differing molecular weight, wherein at least a first portion of the purified proteins is covalently labeled with a mono microperoxidase (MP) N-hydroxy succinimidyl (NHS) ester having the formula:

wherein n is 0-10 and;
R 1 and R 2 are each independently a member selected from the group consisting of hydrogen and an acetyl (Ac), with the proviso that one of R 1 or R 2 is acetyl and one of R 1 or R 2 is hydrogen;
(b) electrophoresing the plurality of proteins to form a separated protein mixture;
(c) transferring the separated protein mixture to a membrane;
(d) adding a chemiluminescent substrate to form a chemiluminescent signal; and
(e) capturing the chemi-signal with a film or an imager.

Assignees

• Li Cor Inc

Inventors

• Padhye Nisha
• Skopp Rose
• Wang Ying

Classifications

• C07K7/06 Primary

  having 5 to 11 amino acids · CPC title

• C12Q1/28

  involving peroxidase · CPC title

• G01N2550/00

  Electrophoretic profiling, e.g. for proteome analysis · CPC title

• G01N2333/908

  acting on hydrogen peroxide as acceptor (1.11) · CPC title

• C07K14/795

  Porphyrin- or corrin-ring-containing peptides · CPC title