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US 2014/0194315 A1Published application
Methods and compositions for rapid functional analysis of gene variants
AssigneeAlbert Einstein College of Medicine of Yeshiva University
InventorsJohnny C. Loke, Harry Ostrer
Life of this patent
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Abstract
Methods and compositions are disclosed for rapid functional analysis of gene variants based on analysis of protein-protein and protein-nucleic acid interactions.
Claims24
A method of multiplex detecting a first protein-second protein interaction, in a sample, for up to at least four distinct first proteins, first proteins A, B, C and D respectively, the method comprising:
- contacting the sample with a (i) a first agent attached to a surface of a magnetic bead that is not labeled with a first primary optically-active label, and (ii) a second primary agent attached to a surface of a magnetic bead that is labeled with a first primary optically-active label, and (iii) a third primary agent attached to a surface of a non-magnetic bead that is not labeled with a second primary optically-active label, and (iv) a fourth primary agent attached to the surface of a non-magnetic bead that is labeled with a second primary optically-active label,
- wherein the first, second, third and fourth primary agents are different agents each capable of capturing the distinct first proteins A, B, C and D, respectively;
- contacting captured first protein-second protein complex(es) with a plurality of secondary agents, each of the plurality being specific for a distinct second protein, and each labeled with a separate secondary optically-active label wherein the secondary optically-active labels are not the same as the primary optically-active labels of the primary agents and are each distinct from the secondary optically-active label of every other of the optically-active labeled secondary agents;
- recovering magnetic beads complexes from the sample by applying a magnetic field;
- recovering non-magnetic bead complexes from the sample based on a non-magnetic physical property of the non-magnetic beads;
- passing the recovered magnetic bead complexes through a flow cytometer or optical plate reader;
- passing the recovered non-magnetic bead complexes through a flow cytometer or optical plate reader;
- detecting the optical signal(s) of the recovered magnetic bead complexes;
- and
- detecting the optical signal(s) of the recovered non-magnetic bead complexes;
- wherein the presence on a magnetic bead complex of only a secondary optically-active label indicates the interaction between the first protein A and a second protein corresponding to the secondary optically-active labeled secondary agent,
- and wherein the presence on a magnetic bead complex of both (i) a first primary optically-active label and (ii) a secondary optically-active label indicates the interaction of the first protein B and a second protein corresponding to the secondary optically-active labeled secondary agent,
- and wherein the presence on a non-magnetic bead complex of only a secondary optically-active label indicates the interaction of the first protein C and a second protein corresponding to the secondary optically-active secondary labeled agent,
- and wherein the presence on a non-magnetic bead complex of both (i) a second primary optically-active label and (ii) a secondary optically-active label indicates the interaction of the first protein D and a second protein corresponding to the secondary optically-active labeled secondary agent.
11 more elements
A method of multiplex detecting protein-nucleic acid interactions in a sample for up to at least four distinct proteins, proteins A, B, C and D respectively, the method comprising:
- a) contacting the sample with a (i) a first agent attached to a surface of a magnetic bead that is not labeled with a first primary optically-active label, and (ii) a second primary agent attached to a surface of a magnetic bead that is labeled with a first primary optically-active label, and (iii) a third primary agent attached to a surface of a non-magnetic bead that is not labeled with a second primary optically-active label, and (iv) a fourth primary agent attached to the surface of a non-magnetic bead that is labeled with a second primary optically-active label,
- wherein the first, second, third and fourth primary agents are different agents each capable of capturing the distinct proteins A, B, C and D, respectively, under conditions which permit capturing to the primary agents a first protein-nucleic acid complex from the sample;
- b) recovering magnetic beads complexes from the sample by applying a magnetic field and recovering non-magnetic bead complexes from the sample based on a non-magnetic physical property of the non-magnetic beads;
- c) contacting one or more of (i) the magnetic bead complexes not having a first primary optically-active label;
- (ii) the magnetic bead complexes having a first primary optically-active label;
- (iii) the non-magnetic bead complexes not having a first primary optically-active label;
- (iv) the non-magnetic bead complexes having a first primary optically-active label, with a Proteinase K so as to digest the proteins thereon and release any nucleic acids bound thereto;
- d) sequencing nucleic acid(s) released in step c)(i) so as to thereby identify the nucleic acids that have interacted with distinct protein A;
- in step c)(ii) so as to thereby identify the nucleic acids that have interacted with distinct protein B;
- in step c)(iii) so as to thereby identify the nucleic acids that have interacted with distinct protein C;
- and/or in step c)(iv) so as to thereby identify the nucleic acids that have interacted with distinct protein D.
8 more elements
12-15. (canceled)
17-18. (canceled)
23-27. (canceled)
(canceled)
A kit for detecting changes in protein expression in cells and for analysis of gene variants, the kit comprising:
- magnetic beads for immunoprecipitation,
- non-magnetic beads for immunoprecipitation,
- a lysis formulation,
- one or more Proteinase K inhibitors,
- one or more phosphatase inhibitors,
- a coupling buffer,
- nucleic acids recovery elution buffer,
- one or more functional variant assay (FVA) buffers,
- a Western loading buffer,
- one or more optically active labels, and
- instructions for use of the kit.
8 more elements
31-36. (canceled)
A kit for obtaining nuclear, cytoplasmic or whole-cell extract from cells or from tissue, the kit comprising:
- cell lysis buffer
- 1M Dithiothreitol (DTT)
- one or more phosphatase inhibitors
- 10×PBS
- one or more phosphatase inhibitors
- 10× hypotonic buffer
- detergent
- written instructions for use of the kit.
5 more elements
Dependencies are inferred from the claim text.