Method for non-radioactive gel shift assays

• US 5,900,358 A
• Filed: 07/24/1997
• Issued: 05/04/1999
• Est. Priority Date: 07/26/1996
• Status: Expired due to Term

• Current Assignee: Linda Besante Ludwig
• Original Assignee: Linda Besante Ludwig
• Inventors: Hughes, Barbara J., Ludwig, Linda Besante
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/899,868
• Time in Patent Office: 649 Days
• Field of Search: 435/6, 436/501, 536/23.1, 536/24.1, 536/24.3-33, 536/25.4, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis

Abstract

A method for a non-radioactive electrophoretic mobility shift assay performed with non-radioactive labeled dsDNA, non-radioactive labeled ssDNA or non-radioactive labeled RNA probes interacting with a nucleic acid binding protein in forming a complex, electrophoresing the mixture containing the complex, transferring the complex to a membrane, and detecting the complex transferred to the membrane by detecting the non-radioactive label in the complex.

Claims

1. A method for a electrophoretic gel mobility shift assay comprising

(a) contacting in a mixture a nucleic acid binding protein with a non-radioactive labeled nucleic acid molecule comprising a molecular probe under suitable conditions to promote specific binding interactions between the protein and the probe in forming a complex, wherein said probe is selected from the group consisting of dsDNA, ssDNA, and RNA;

(b) electrophoresing the mixture;

(c) transferring, using positive pressure blot transfer or capillary transfer, the complex to a membrane, wherein the membrane is positively charged nylon; and

(d) detecting the complex bound to the membrane by detecting the non-radioactive label in the complex.

2. The method of claim 1, wherein the non-radioactive label is selected from the group consisting of a fluorescent molecule, a chemiluminescent molecule, and biotin.

3. The method of claim 2, wherein the non-radioactive label is a fluorescent molecule.

4. The method of claim 2, wherein the non-radioactive label is a chemiluminescent molecule.

5. The method of claim 2, wherein the non-radioactive label is biotin.

6. The method of claim 1, wherein the probe is dsDNA.

7. The method of claim 1, wherein the probe is ssDNA.

8. The method of claim 1, wherein the probe is RNA.