Protein fractionation

• US 7,172,739 B2
• Filed: 05/22/2002
• Issued: 02/06/2007
• Est. Priority Date: 05/22/2001
• Status: Expired due to Fees

• Current Assignee: University of Vermont & State Agricultural College
• Original Assignee: University of Vermont & State Agricultural College
• Inventors: Maughan, David W.
• Primary Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US10/154,443
• Publication Number:
  US 20020192759A1
• Time in Patent Office: 1,721 Days
• Field of Search: 435/91.1, 435/40.5, 435/40.52, 514/12, 210/23, 422/276, 422/255, 422/261
• US Class Current: 422/276
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types

Abstract

A method and apparatus for physically separating polypeptide constituents of a sample fluid by (a) providing a sample fluid comprising a mixture of polypeptides of differing physical and/or chemical properties; (b) contacting the fluid with at least two individual polymer matrix units, each polymer matrix unit preferentially accepting a different set of polypeptides on the basis of one or more physical and/or chemical properties; and (c) extracting the set of polypeptides from each polymer matrix unit.

Claims

1. A method for fractionating a tissue sample to separate a fraction of cellular components from the tissue sample, the method comprising:

(a) providing a perforated perfusion chamber;

(b) placing a tissue sample in the chamber, wherein the sample is held in the chamber;

(c) exposing the tissue sample in the chamber to a fractionating solution by;

(i) flowing the fractionating solution into the chamber though and/or around the sample at a rate calculated to permit fractionation of the sample;

or

(ii) flowing the fractionating solution into the chamber and maintaining the solution in the chamber for a time sufficient to permit fractionation of the sample,

(d) concurrently with (c)(i) or following (c)(ii), flowing the fractionating solution out of the chamber to form a bubble or droplet of solution or an array of bubbles or droplets under oil, the fractionating solution comprising the fraction of the cellular components of the tissue sample, wherein the remainder of the tissue sample is held in the chamber.

2. The method of claim 1 wherein the tissue sample has a mass which is less than 1 mg.

3. The method of claim 1 wherein the tissue sample of has a mass which is less than 0.1 mg.

4. The method of claim 1 further comprising enriching polypeptide content of the fractionating solution after it leaves the chamber by removing water therefrom.

5. The method of claim 4 wherein the water is removed using a polymeric matrix unit which absorbs water without absorbing polypeptides.

6. The method of claim 1 wherein steps 1(c) and 1(d) are repeated using fractionation solutions which extract different cellular components.

7. The method of claim 1 wherein one or more of the steps is automated.

8. The method of claim 1 wherein one or more of the steps is performed robotically.

9. The method of claim 1 wherein steps 1(c) and 1(d) are repeated using two or more fractionating solutions selected from the group consisting of:

(a) a fractionating solution which solubilizes plasma membrane while leaving membranes of intracellular organelles intact, permitting cytosolic polypeptides to diffuse out of cells of the tissue sample and to be separated from non-cytosolic cellular material;

(b) a fractionating solution which solubilizes membranes of organelles, permitting organellar polypeptides to diffuse out of cells of the tissue sample and to be separated from non-organellar cellular material; and

(c) a fractionating solution which solubilizes cytomatrix polypeptides permitting cytomatrix polypeptides to be separated from non-cytomatrix cellular material.

10. The method of claim 9 wherein the fractionating solution of 9(a) comprises a detergent component.

11. The method of claim 10 wherein the detergent component comprises saponin.

12. The method of claim 9 wherein the solution of 9(b) comprises a detergent.

13. The method of claim 12 wherein the detergent component comprises triton X100.

14. The method of claim 9 wherein the solution of 9(c) comprises urea.