Formulations and methods for denaturing proteins
Reagents, methods and kits for denaturation of protein are provided.
1. A formulation for denaturing proteins comprising:
a lithium salt at a concentration of about 2.5–4.0 M,
an alcohol at a concentration of about 25–40% v/v, and
a citrate at a concentration of about 25–100 mM.
2. The formulation of claim 1 , which formulation lacks EDTA.
3. The formulation of claim 1 , wherein the formulation lacks a chaotropic substance.
4. The method of claim 3 , wherein the chaotropic substance is guanidinium salt, urea, ammonium, cesium, rubidium, potassium, or iodide salt.
5. The formulation of claim 1 , wherein the lithium salt is lithium chloride or lithium bromide.
6. The formulation of claim 1 , wherein the lithium salt is lithium chloride.
7. The formulation of claim 1 , wherein the lithium salt is at a concentration of about 3.5 M.
8. The formulation of claim 1 , wherein the alcohol is ethanol or methanol.
9. The formulation of claim 1 , wherein the alcohol is ethanol.
10. The formulation of claim 1 , wherein the alcohol is at a concentration of about 30% alcohol.
11. The formulation of claim 1 , wherein the citrate is trisodium citrate.
12. The formulation of claim 1 , wherein the citrate is at a concentration of about 50 mM.
13. The formulation of claim 1 , wherein the formulation has a pH between about 6 and about 8.
14. The formulation of claim 1 , wherein the solution has a pH between about 7.0 and about 7.5.
15. A method for denaturing protein from a solid support, comprising contacting the solid support with the formulation of claim 1 , such that protein present on the solid support is denatured.
16. The method of claim 15 , wherein the protein is an enzyme.
17. The method of claim 16 , wherein the enzyme is a DNase.
18. The method of claim 17 , wherein the DNase is DNase I.
19. The method of claim 15 , wherein the solid support comprises components of silica, cellulose, cellulose acetate, nitrocellulose, nylon, polyester, polyethersulfone, polyolefin, or polyvinylidene fluoride, or combinations thereof.
20. The method of claim 15 , wherein the solid support is contained in a vessel, wherein the vessel is a centrifuge tube, spin tube, syringes, cartridge, chamber, multiple-well plate, or test tube, or combinations thereof.
21. A method for purifying substantially pure and undegraded RNA from biological material comprising RNA, comprising the steps of:
(a) contacting a solid support with the formulation of claim 1 , such that protein present on the solid support is denatured;
(b) contacting the solid support with a sample comprising RNA such that the RNA binds to the solid support;
(c) washing the solid support with a series of wash solutions to remove biological materials other than bound RNA, wherein the series of wash solutions comprises a first wash containing alcohol and an RNA-complexing salt at a concentration of at least 1M and a second wash containing an alcohol, buffer and an optional chelator; and
(d) preferentially eluting the bound RNA from the solid support with an RNA elution solution in order to obtain substantially pure RNA.
22. The method of claim 21 , wherein the solid support is contained in a vessel, wherein the vessel is a centrifuge tube, spin tube, syringe, cartridge, chamber, multiple-well plate, test tube, or combination thereof.
23. The method of claim 21 , wherein the RNA-complexing salt is lithium chloride.