Methods and compositions for dual extraction of protein and nucleic acid
View Patent ↗The present invention provides a method of isolating nucleic acid and protein from the same biological sample, comprising, in the following order: a) disrupting the biological sample; b) contacting the disrupted biological sample of (a) with a protein lysis buffer that lacks any component that denatures or reduces protein to produce a first lysate; c) centrifuging the first lysate of (b) to produce a first supernatant containing protein and a pellet containing nucleic acid; d) removing the first supernatant of (c), thereby isolating protein from the biological sample; e) contacting the pellet of (d) with nucleic acid lysis buffer to produce a second lysate; f) centrifuging the second lysate of (e) to produce a second supernatant containing nucleic acid; and g) removing the second supernatant of (f), thereby isolating nucleic acid from the same biological sample.
1. A method of isolating nucleic acid and undenatured and unreduced protein from the same biological sample, comprising, in the following order:
a) disrupting the biological sample, which comprises protein that is not denatured and not reduced;
b) contacting the disrupted biological sample of (a) with a protein lysis buffer that lacks any component that denatures or reduces protein to produce a first lysate;
c) centrifuging the first lysate of (b) to produce a first supernatant containing undenatured and unreduced protein and a pellet containing nucleic acid;
d) removing the first supernatant of (c), thereby isolating undenatured and unreduced protein from the biological sample;
e) contacting the pellet of (d) with nucleic acid lysis buffer to produce a second lysate;
f) centrifuging the second lysate of (e) to produce a second supernatant containing nucleic acid; and
g) removing the second supernatant of (f), thereby isolating nucleic acid from the same biological sample.
2. The method of claim 1 , wherein the protein lysis buffer of (b) is Protein Lysis Buffer A (phosphate buffered saline with 0.05% Tween-20) or BB-PVP-Tw buffer [borate buffer 0.1M, pH 7.5, 0.2% polyvinylpyrrolidone (PVP), 0.5% Tween-20].
3. The method of claim 1 , wherein the protein lysis buffer of (b) lacks sodium dodecyl sulfate (SDS), urea, guanidine, 2-mercaptoethanol and dithiothreitol.
4. The method of claim 1 , wherein the nucleic acid lysis buffer of (e) is PA lysis buffer (4M guanidine thiocyanate, 10 mM Tris, pH 7.5).
5. The method of claim 1 , further comprising the step of detecting protein in the first supernatant of (d) by ELISA.
6. The method of claim 1 , further comprising the step of analyzing the nucleic acid in the second supernatant of (g) by an amplification reaction.
7. The method of claim 6 , wherein the amplification reaction is a polymerase chain reaction (PCR).
8. The method of claim 1 , wherein the biological sample is from a plant.
9. The method of claim 8 , wherein the biological sample is leaf tissue from the plant.
10. The method of claim 8 , wherein the plant is corn, soy, sugarcane or wheat.
11. The method of claim 1 , carried out in a high throughput format.