METHOD AND MEANS FOR AN ISOLATION OF MEMBRANE-BOUND PROTEINS FROM A BIOLOGICAL SAMPLE, PREFERABLY PROCESSED PLANT SEED MEAL

Abstract

The present invention provides the method and means for an efficient isolation of membrane-bound proteins from biological samples, e.g. in samples from raw or processed plant material, preferably defatted plant seed meal such as canola meal. The biological sample can be highly processed, e.g. by applying high temperature, pressure, or a chemical treatment and can be derived from seed matrices as well as other typical plant tissues for example seed, grain, leaf, root, or pollen. The invention comprises the provision of a novel extraction buffer (MEB) and its application in the method of the invention, wherein the buffer has a strong alkaline pH of 10 to 12.5 and comprises a soluble concentration of detergent at a level of 0.5% to 5%.

Claims

1. Method for the extraction of membrane-bound proteins from a plant sample comprising: Incubating the sample at in a buffering reagent of pH 10 to 12.5 comprising a soluble concentration of detergent at a level of 0.5% to 5% (membrane extraction buffer (MEB)) and wherein the buffer MEB comprises a reducing agent.

2. The method of claim 1 applying the lysis buffer MEB at a pH 11.5 to 12.5.

3. The method of claim 1, incubating the sample at an incubation temperature of 35 C. to 55 C.

4. The method of claim 1 comprising applying the lysis buffer MEB with a soluble concentration of an ionic detergent of 1.5 to 5%.

5. The method of claim 1 comprising applying a lysis buffer MEB comprising 1.5 to 3% LDS.

6. (canceled)

7. The method of claim 1, wherein the lysis buffer MEB comprises DTT, BME or TCEP-HCL.

8. The method of claim 1, wherein the lysis buffer MEB comprises one or more reducing agents equivalent to 20 mM to 70 mM TCEP.

9. The method of claim 1, wherein the MEB buffer comprises 20 mM to 70 mM TCEP, or 30 to 120 nM DTT or 1 to 5% BME.

10. The method of claim 1 comprising a pH dependent buffering agent, or a salty solution.

11. The method of claim 10, wherein the buffering agent is Tris-CL, HEPES, CAPS, and/or Sodium Phosphate buffer NaH2P04/Na2HP04.

12. The method of claim 10, wherein the buffering agent is i. 5 to 300 mM Tris-CL, ii. 20 to 40 mM HEPES, iii. 10 to 40 mM Sodium phosphate (NaH2P04/Na2HP04), and/or iv. 50 mM to 500 mM CAPS.

13. The method of claim 1 comprising a viscosity agent.

14. The method of claim 1 comprising a viscosity agent, wherein the viscosity agent is 10% to 25% glycerol.

15. The method of claim 1, comprising a buffering reagent at pH 10 to 12.5 a LDS content of 1.5% to 2.5%, and a reducing reagent 20 to 70 mM TCEP, wherein the buffering reagent is 100 mM to 150 mM Tris-Cl and 10% to 20% glycerol.

16. The method of claim 1, wherein the incubation time at the incubation temperature is between 10 min and 180 min.

17. The method of claim 1, wherein extraction is performed as a multiple sequential extraction.

18. (canceled)

19. The method of claim 1, wherein the tissue is derived from a leave, seed, grain, leaf, root, or pollen.

20. The method of claim 1, wherein the material is defatted plant seed meal.

21. The method of claim 1, comprising the following steps: a. Incubating the sample at a temperature of 35 C. to 55 C. in a lysis buffer MEB comprising a detergent and a reducing agent, b. extracting the membrane-bound proteins, c. diluting the extract to adjust the pH for the analysis of the proteins d. analyzing the proteins without prior precipitation, resuspension or solubilization.

22. The method of claim 1, wherein the extracted proteins are analyzed in, Western blotting, mass spectrometry, Isoelectric focusing, or 2D protein separation.

23. A composition comprising a buffering reagent at pH 10 to 12.5, and a detergent content of 0.5% to 2.5% and a reducing agent.

24. The composition of claim 23 comprising at pH10.0 to pH 12.5: i. 1.5% to 2.5% LDS, and ii. 30 mM to 70 mM TCEP.

25. The composition of claim 23 further comprising tissue and/or membrane derived from an organism.

26. The composition of claim 23 having a temperature of 35 C. to 55 C.

27. The method of claim 1, comprising one or more of the following steps: a. Grinding the sample b. Cooling the sample on dry ice c. Weighing the sample d. Extracting polypeptides from the grinded and cooled sample by applying an extraction buffer MEB. e. Spinning the sample and transferring the supernatant to a new test tube, whereby the test tube is cooled.

28. The method of claim 1, comprising a. Providing plant meal, b. Adding the prewarmed an extraction buffer MEB, c. Grinding the sample, d. Incubating the sample at for 5 to 15 min, at 30 C. to 45 C., e. Centrifuging the sample and transferring the supernatant to a new container, and f. Analyzing the supernatant.

29. The method of claim 1, comprising the quantitative analysis of the one or more polypeptides by applying one or more antibody specifically binding to a potentially membrane-bound polypeptide.

30. The method of claim 1, wherein the polypeptide is a desaturase or elongase, preferably a transgenic desaturase or elongase.

Description

EXAMPLES

Examples 1

General Extraction and Sample Preparation Procedure

[0097] Extraction buffer components (120 mM Tris, 20% glycerol, 2% LDS, and 50 mM TCEP at pH 12.0) were combined and final pH adjusted to approximately 12.0. The solution was warmed to 40 C. to improve solubility of LDS. Homogenized canola meal samples (10-50 mg) were extracted by combining an appropriate ratio of canola meal tissue to warm extraction buffer (for example 1:40-50 for included experiments) and a stainless-steel bead into a 2 mL screw cap tube. The tube containing the sample, extraction buffer, and bead were vigorously shaken for 2 minutes at 1200 rpm using a Geno/Grinder (SPEX Sample Prep, Metuchen, N.J.). The resulting sample lysate was incubated at 40 C. with vibration using an Eppendorf Thermomixer for 30 minutes. Following incubation, the lysate was centrifuged at 12,000 to 16,000 rcf for 5-15 minutes. The supernatant was aspirated and transferred to a new polyproylene tube. The supernatant was diluted as needed using extraction buffer (for example 24-fold to 60-fold for included experiments), then prepared for analysis using the ProteinSimple capillary western blot platform. Sample preparation was performed following the manufacturer's instructions provided with kit reagents. Primary antibodies specific to membrane-bound proteins 1, 2, and, 3 were utilized to detect their respective protein targets.

[0098] Quantification of extracted protein was interpolated using a standard curve prepared by fortifying extraction buffer with homologously-expressed reference standard protein. The reference standards were diluted and prepared for analysis following the same procedure as the tissues samples.

Examples 2

pH Effects on Extractability

[0099] The effects of extraction buffer pH on protein extractability from canola meal were determined by extracting three unique meal samples with buffers prepared at pH 8.0, 10.0, and 12.0. The general extraction and sample preparation procedure was otherwise followed. Extractability of the sample was determined by comparing the signal response of Protein 1 extracted with pH 12 and pH 10 buffer to the signal response of the protein extracted with pH 8 buffer.

Examples 3

Effects of Detergent and Reducing Agent on Extractability

[0100] The effects of reducing agent (TCEP) and detergent (LDS) components were evaluated by performing extractions of Protein 1 without one of these components and comparing these results to the extractability achieved using the optimized extraction buffer including all components described in the general extraction and sample preparation procedure.

Examples 4

[0101] Extractability of Membrane-Bound Proteins 1, 2, and 3 and Soluble Protein 4 from Canola Meal

[0102] The extractability of membrane-bound proteins and a soluble protein from canola meal was demonstrated by extracting three unique proteins 1, 2, and 3 using the general extraction and sample preparation procedure. The level of each protein was measured from three successive extractions of transgenic (containing proteins 1, 2, and 3) canola meal samples. Extractability of each protein was expressed as the measured concentration of the protein from the first extraction with respect to the sum of the measured concentrations from all three extractions.

Example 5

Standard Extraction Buffer:

[0103] RIPA: 20 mM TrisCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP40, 1% Sodium deoxycholate, Protease/phosphatase inhibitors cocktail

[0104] Cell Lysis Buffer: 20 mM TrisCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, Protease/phosphatase inhibitors cocktail

[0105] SDS Lysis Buffer: 50 mM TrisCl pH 8.1.10 mM EDTA, 1% SDS

[0106] IP Lysis buffer: 25 mM TrisCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40.5% glycerol

[0107] T-Per: 25 mM Bicine pH 7.6, 150 mM NaCl, Proprietary detergent

[0108] Cellytic MT: Bicine, unknown concentration, 150 mM NaCl, proprietary dialysable mild detergent Commercial lysates prepared in various lysis buffers: 62.5 mM TrisCl, pH 6.8, 2% SDS, 10% Glycerol.