Advancing 2-DE Techniques: High-Efficiency Protein Extraction From Lupine Roots
推进二维电泳技术：高效提取羽扇豆根蛋白
发布: 2025年10月05日第15卷第19期 DOI: 10.21769/BioProtoc.5461 浏览次数: 143
评审: Nicolás M. CecchiniSamik Bhattacharya
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Abstract
Protein isolation combined with two-dimensional electrophoresis (2-DE) is a powerful technique for analyzing complex protein mixtures, enabling the simultaneous separation of thousands of proteins. This method involves two distinct steps: isoelectric focusing (IEF), which separates proteins based on their isoelectric points (pI), and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins by their relative molecular weights. However, the success of 2-DE is highly dependent on the quality of the starting material. Isolating proteins from plant mature roots is challenging due to interfering compounds and a thick, lignin-rich cell wall. Bacterial proteins and metabolites further complicate extraction in legumes, which form symbiotic relationships with bacteria. Endogenous proteases can degrade proteins, and microbial contaminants may co-purify with plant proteins. Therefore, comparing extraction methods is essential to minimize contaminants, maximize yield, and preserve protein integrity. In this study, we compare two protein isolation techniques for lupine roots and optimize a protein precipitation protocol to enhance the yield for downstream proteomic analyses. The effectiveness of each method was evaluated based on the quality and resolution of 2-DE gel images. The optimized protocol provides a reliable platform for comparative proteomics and functional studies of lupine root responses to stress, e.g., drought or salinity, and symbiotic interactions with bacteria.
Key features
• Protocol tailored for isolating proteins from lupine roots, including those involved in symbiotic relationships with bacteria.
• Our method is suitable for analyzing complex protein mixtures through IEF and SDS-PAGE for high-resolution separation.
• Optimized precipitation method increases protein yield for downstream mass spectrometry and comparative proteomic analyses.
Keywords: Protein isolation (蛋白分离)
Protein precipitation (蛋白沉淀)
Yellow lupine (黄羽扇豆)
Two-dimensional gel electrophoresis (二维凝胶电泳)
Optimization (方法优化)
Background
Proteomic analyses are crucial in plant physiology research, providing valuable insight into plant function. However, these studies' success heavily depends on the quantity and quality of the extracted material. Proteins play key roles in enzymatic activity, signaling, and defense, directly influencing plant health and its interactions with the surrounding ecosystem. This is especially significant for roots, which are essential for nutrient uptake and transport. Despite their importance, isolating proteins from mature roots presents several technical challenges that limit progress in proteomic and biochemical analyses. Traditional protein isolation protocols, typically designed for leaf or seed tissues, are often ineffective for roots, due to their distinct biochemical composition, unique physiology, and lower protein content compared to aboveground tissues [1]. Therefore, it becomes necessary to adapt homogenization methods, select appropriate extraction buffers containing protease inhibitors, and optimize purification techniques.
One of the major challenges in isolating proteins from plant roots is the presence of phenolic compounds, which, upon oxidation, form stable complexes with proteins, thereby reducing their recovery [2,3]. Other significant issues include the increased rigidity of the cell walls, which impede homogenization, as well as the presence of polysaccharides and other carbohydrates that increase sample viscosity. Additionally, certain compounds such as detergents, salts, or lipids may interfere with protein extraction and co-precipitate with the proteins of interest [4,5]. Root tissue is also characterized by high endogenous proteolytic activity, which can lead to rapid protein degradation during homogenization and subsequent extraction steps [3,4]. This degradation is especially pronounced when cell structures are disrupted, causing proteases to be released from their native compartments. To mitigate this, the use of protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) or commercially available inhibitor mixtures, is critical. In legumes, where specific bacterial proteases are present due to a symbiotic relationship with Rhizobium bacteria, additional cysteine protease inhibitors may be required [6]. Furthermore, the entire procedure should be conducted at the lowest possible temperature to reduce enzymatic activity, and the protein isolation process should be performed as quickly as possible to minimize the exposure of proteins to proteases [3,4]. In some cases, pre-purification of samples using trichloroacetic acid (TCA)/acetone protein precipitation can be beneficial, as it helps to inactivate certain proteolytic enzymes. This method, originally developed by Damerval et al. [7] and later modified by Saravanan and Rose [8], Isaacson et al. [3], and Fic et al. [9], is widely applied to various tissues. It is important to note that prolonged exposure of the extract to acetone can negatively affect protein structure, which may, in turn, compromise the result of MS/MS analysis at later stages [10]. Additionally, protein degradation can occur due to prolonged exposure to low pH [4,11]. To mitigate this, an aqueous TCA solution is often used for precipitation [12], although the resulting proteins may be more challenging to solubilize.
Given the critical role of proteomic research in understanding plant physiology and the stringent requirements regarding the quantity and purity of protein material, it is essential to continually adapt methods to meet the specific needs of the plant tissue being studied. This is especially important for economically significant species, such as yellow lupine (Lupinus luteus L.), where the quality and yield of protein extraction directly impact research outcomes and practical applications. In the context of the European Union’s pro-environmental policies, this species is recognized as a natural source of nitrogen (N2), fixed from the atmosphere through symbiosis with Bradyrhizobium bacteria. This process reduces the need for synthetic fertilizers, thereby minimizing environmental impact. Additionally, N2 fixed in this way is stored in the soil, making it less prone to leaching by precipitation. Studying the proteome offers valuable insight into these processes, enabling the identification of proteins involved in the plant's response to various environmental conditions. This research will advance our current understanding of plant N2 fixation and its broader ecological implications. The challenge of isolating proteins from yellow lupine roots was previously addressed by Orzoł and Piotrowicz-Cieślak [13] through their study focused on seedlings at early developmental stages up to 12 days. The authors performed analyses on young plants that had not yet developed nodules and contained lower levels of secondary metabolites, which could affect isolation efficiency. This study aims to compare two commonly used protein isolation methods for obtaining root material from lupine suitable for two-dimensional electrophoresis. Additionally, we have optimized a protein precipitation method to increase the yield of material for proteomic analyses. The effectiveness of each method was assessed by comparing 2-DE PAGE gel images of the resulting protein samples. Our optimization assay identified the Tris-EDTA protocol as the most effective protein isolation method, with a 1-h TCA/acetone precipitation step yielding the highest protein recovery.
Materials and reagents
Biological materials
1. Commercial yellow lupine cultivar Taper was grown in phytotron chambers under controlled conditions, as optimized previously by Frankowski et al. [14]. In brief, seeds were sourced from Poznań Plant Breeding Tulce (Wiatrowo, Poland), treated with Sarfun (2.5 mL per kg of seeds; Organika-Sarzyna S.A., Nowa Sarzyna, Poland), and inoculated for 2 h with Nitragina (3 g per kg of seeds, BIOFOOD S.C., Wałcz, Poland). Seeds were then sown into pots filled with class V field soil (53° 6' 22.383" N 18° 27' 1.922" E) and grown at 22 °C ± 1 °C under long day conditions (16 h light/8 h dark, 110 μmol m-2 s-1 cool white fluorescent lighting; Polam, Warsaw, Poland). On day 48 of cultivation, roots were harvested as described by Burchardt et al. [15]. The root system was divided into two distinct zones: an upper region bearing nitrogen-fixing nodules, and a lower, nodule-free region. For this study, nodules were removed from the upper root segment, and the remaining tissue was frozen in liquid nitrogen and stored at -80 °C for further analysis.
Reagents
1. Phenol solution, BioReagent, equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA), for molecular biology (Sigma-Aldrich, Merck KGaA, catalog number: P4557)
2. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, Merck KGaA, catalog number: 11667289001)
3. Sucrose, molecular biology grade (Milipore, Merck KGaA, catalog number: 573113)
4. β-Mercaptoethanol (Sigma-Aldrich, Merck KGaA, catalog number: M6250)
5. Trizma® base, ≥99.9% (titration), crystalline (Sigma-Aldrich, Merck KGaA, catalog number: T4661)
6. Hydrochloric acid (HCl) 37%, ACS reagent (Sigma-Aldrich, Merck KGaA, catalog number: 258148)
7. PMSF (Roche, F. Hoffmann-La Roche AG, catalog number: PMSF-RO)
8. Ammonium acetate (Chempur, POL-AURA, catalog number: 111392705)
9. Methanol, anhydrous for analysis (max. 0.003% H2O) (Supelco, Merck KGaA, catalog number: 106012)
10. Acetone suitable for HPLC, ≥99,9% (Sigma-Aldrich, Merck KGaA, catalog number: 270725)
11. Trichloroacetic acid (TCA), ACS reagent (Sigma-Aldrich, Merck KGaA, catalog number: T6399)
12. Ethyl alcohol, pure, ≥99.5%, ACS reagent, 200 proof (Sigma-Aldrich, Merck KGaA, catalog number: 459844)
13. EDTA (Sigma-Aldrich, Merck KGaA, catalog number: E6758)
14. NaCl pure p.a. (Avantor Performance Materials Poland S.A., catalog number: PA-06-794121116)
15. Urea, powder, BioReagent, for molecular biology, suitable for cell culture (Sigma-Aldrich, Merck KGaA, catalog number: U5378)
16. Thiourea, ACS reagent, ≥99.0% (Sigma-Aldrich, Merck KGaA, catalog number: T8656)
17. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Roche, F. Hoffmann-La Roche AG, catalog number: CHAPS-RO)
18. Bovine serum albumin (BSA), lyophilized powder, BioReagent, suitable for cell culture (Sigma-Aldrich, Merck KGaA, catalog number: A9418)
19. Bradford reagent (Supelco, Merck KGaA, catalog number: B6916)
20. Ammonium persulfate (APS), BioXtra, ≥98.0% (Sigma-Aldrich, Merck KGaA, catalog number: A7460)
21. Acetic acid 99.5%–99.9% PURE (Avantor Performance Materials Poland S.A., catalog number: PA-06-568760114)
22. Coomassie Brilliant Blue R-250 protein stain powder (Bio-Rad Laboratories, Inc., catalog number: 1610400)
23. 30% Acrylamide/Bis Solution, 29:1 (Bio-Rad Laboratories, Inc., catalog number: 1610156)
24. N,N,N′,N′-tetramethylethylenediamine (TEMED) (Sigma-Aldrich, Merck KGaA, catalog number: T9281)
25. Glycine (Sigma-Aldrich, Merck KGaA, catalog number: G7126)
26. Protein molecular weight marker PageRulerTM Prestained Protein Ladder, 10–180 kDa (Thermo Fisher Scientific Inc., catalog number: 26617)
27. ReadyPrep 2-D Starter Kit 2-D Gel Electrophoresis kit (Bio-Rad Laboratories, Inc., catalog number: 1632105)
28. ReadyStrip IPG strips 7 cm, pH 3–10 nonlinear (Bio-Rad Laboratories, Inc., catalog number: 1632002)
29. Mineral oil (Bio-Rad Laboratories, Inc., catalog number: 1632129)
Solutions
A. For the phenol method
1. 100 mM Tris-HCl, pH 8.0 (see Recipes)
2. SDS buffer (see Recipes)
3. 100 mM ammonium acetate in methanol (see Recipes)
4. Anhydrous methanol containing 0.1% (w/v) ammonium acetate (see Recipes)
5. 80% (v/v) acetone (see Recipes)
B. For the modified Tris-EDTA method
1. 50 mM Tris-HCl buffer, pH 7.5 (see Recipes)
2. 20% (w/v) TCA in acetone (see Recipes)
3. 20% (w/v) TCA in H2O (see Recipes)
4. 1:1 (v/v) mixture of ethanol and ethyl acetate (see Recipes)
C. Common solutions
1. 1.5 M Tris-HCl, pH 8.8 (see Recipes)
2. 10% (w/v) SDS (see Recipes)
3. 10% (w/v) APS (see Recipes)
4. 7 M urea, 2 M thiourea, 4% (w/v) CHAPS (see Recipes)
5. Bradford assay (see Recipes)
6. 12% (w/v) polyacrylamide gel (see Recipes)
7. 1× SDS-PAGE running buffer (see Recipes)
8. Gel staining solution (see Recipes)
9. Gel destaining solution (see Recipes)
Recipes
A. For the phenol method (based on Wang et al. [4] with modifications of Nisar et al. [16])
1. 100 mM Tris-HCl, pH 8.0
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Trizma base | 100 mM | 1.21 g |
| H2O (MilliQ) | n/a | up to 100 mL |
a. Weigh the appropriate amount of Trizma base and transfer it to a bottle.
b. Add 70 mL of H2O (MilliQ) and insert a stirring rod in a bottle.
c. Place the bottle on a magnetic stirrer set to approximately 400 rpm and stir until the Trizma base is fully dissolved.
d. Then insert the pH meter electrode and, while stirring, gradually add HCl in small portions to adjust the pH to 8.0.
e. After reaching the desired pH, dilute the solution with H2O (MilliQ) to a final volume of 100 mL and mix thoroughly. Store the prepared solution at 4 °C for up to 4 weeks.
2. SDS buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| SDS | 2% | 2 g |
| Sucrose | 30% | 30 g |
| PMSF | 1 mM | 17.42 mg |
| β-mercaptoethanol | 5% | 5 mL |
| 100 mM Tris-HCl, pH 8.0 | 100 mM | up to 100 mL |
a. Weigh the appropriate amounts of the ingredients.
b. Since PMSF is insoluble in water, dissolve it in approximately 5 mL of ethanol, methanol, or isopropanol.
c. Transfer all ingredients to a bottle and add 70 mL of 100 mM Tris-HCl, pH 8.0.
d. Mix until all components are dissolved, then add 5 mL of β-mercaptoethanol. Adjust the volume to 100 mL with 100 mM Tris-HCl, pH 8.0, and mix thoroughly. Store the buffer at 4 °C for up to 4 weeks.
3. 100 mM ammonium acetate in methanol
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Ammonium acetate | 100 mM | 1.54 g |
| Anhydrous methanol | 100% | up to 200 mL |
a. Weigh 1.5416 g of ammonium acetate and transfer it to a bottle.
b. Add 150 mL of anhydrous methanol and mix thoroughly.
c. Adjust the volume to 200 mL with methanol and mix again. Store the solution at 4 °C for up to 4 weeks.
4. Anhydrous methanol with 0.1% (w/v) ammonium acetate
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Ammonium acetate | 0.1% | 100 mg |
| Anhydrous methanol | 100% | up to 100 mL |
a. Weigh 100 mg of ammonium acetate and transfer it to a bottle.
b. Add 80 mL of anhydrous methanol and mix thoroughly until fully dissolved.
c. Adjust the volume to 100 mL with methanol and mix again.
d. Store the solution at 4 °C for up to 4 weeks.
5. 80% (v/v) acetone
| Reagent | Final concentration | Volume |
|---|---|---|
| Acetone | 80% | 80 mL |
| H2O (MilliQ) | n/a | up to 100 mL |
a. Measure 80 mL of acetone in a cylinder, then add H2O (MilliQ) to bring the total volume to 100 mL.
b. Transfer the solution to a bottle and mix thoroughly.
c. Store at room temperature for up to 6 months.
B. For the modified Tris-EDTA method (based on Azri et al. [17])
1. 50 mM Tris-HCl buffer, pH 7.5
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Trizma base | 50 mM | 605.7 mg |
| EDTA | 2 mM | 58.45 mg |
| PMSF | 1 mM | 17.42 mg |
| H2O (MilliQ) | n/a | up to 100 mL |
a. Weigh all required ingredients and transfer them to a clean bottle.
b. As PMSF is not water-soluble, dissolve the measured amount in 5 mL of ethanol.
c. Add 70 mL of H2O (MilliQ) to the bottle and place the solution on a magnetic stirrer.
d. Adjust the pH to 7.5 using HCl while stirring.
e. Once the pH is stabilized, top up the volume to 100 mL with H2O (MilliQ) and mix thoroughly.
f. Store the solution at 4 °C for up to 3 weeks.
2. 20% (w/v) TCA in acetone
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| TCA | 20% | 20 g |
| Acetone | 100% | up to 100 mL |
a. Weigh the required amount of TCA and dissolve it in 50 mL of acetone.
b. Mix thoroughly until fully dissolved.
c. Adjust the final volume to 100 mL with acetone and mix again.
d. Store the solution at 4 °C for up to 4 weeks.
3. 20% (w/v) TCA in H2O
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| TCA | 20% | 20 g |
| H2O (MilliQ) | n/a | up to 100 mL |
a. Weigh out the required amount of TCA and dissolve it in 50 mL of H2O (MilliQ).
b. Mix thoroughly until fully dissolved.
c. Adjust the final volume to 100 mL with H2O (MilliQ) and mix again.
d. Store the solution at 4 °C for up to 4 weeks.
4. 1:1 (v/v) mixture of ethanol and ethyl acetate
| Reagent | Final concentration | Volume |
|---|---|---|
| Ethanol | 50% | 100 mL |
| Ethyl acetate | 50% | 100 mL |
a. Measure 100 mL of 100% ethanol and 100 mL of 100% ethyl acetate.
b. Transfer both liquids into a clean bottle and mix thoroughly by swirling.
c. Store the solution at 4 °C for up to 4 weeks.
C. Common solutions
1. 1.5 M Tris-HCl, pH 8.8
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Trizma base | 1.5 M | 18.171 g |
| H2O (MilliQ) | n/a | up to 100 mL |
a. Weigh Trizma base, and dissolve in 80 mL of H2O (MilliQ) in a sterile bottle.
b. Add a magnetic stir bar to the bottle and place it on a magnetic stirrer plate.
c. Set the stirring speed to approximately 800 rpm.
d. Once Trizma base is completely dissolved, adjust the pH to 8.8 with HCl.
e. Dilute the solution to a final volume of 100 mL with water (MilliQ).
f. Store the prepared solution at 4 °C for up to 4 weeks.
2. 10% (w/v) SDS
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 10% SDS | 10% | 1 g |
| H2O (MilliQ) | n/a | up to 10 mL |
a. Weigh the required amount of SDS and dissolve it in 5 mL of H2O (MilliQ) in a sterile bottle.
b. Mix thoroughly by swirling until the SDS is completely dissolved.
c. Once dissolved, adjust the final volume to 10 mL with water (MilliQ). Store the prepared solution at room temperature for up to 6 months.
3. 10% (w/v) APS
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| APS | 10% | 1 g |
| H2O (MilliQ) | n/a | up to 10 mL |
a. Weigh 1 g of APS and dissolve it in 5 mL of H2O (MilliQ) in a sterile bottle.
b. Mix gently by swirling until the crystals are completely dissolved.
c. After that, adjust the final volume to 10 mL with water (MilliQ).
d. Aliquot the solution into 2 mL Eppendorf tubes and store at -20 °C for up to 6 months.
4. 7 M urea, 2 M thiourea, 4% (w/v) CHAPS
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Urea | 7 M | 12.61 g |
| Thiourea | 2 M | 4.56 g |
| CHAPS | 4% | 1.2 g |
| H2O (MilliQ) | n/a | up to 30 mL |
a. Weigh the appropriate amount of reagents and dissolve them in 15 mL of H2O (MilliQ) in a sterile bottle.
b. Place a magnetic stirrer in the bottle and position it on a magnetic stirrer plate.
c. Set the stirring speed to approximately 800 rpm. Optional heating may be applied, but care should be taken to ensure that the temperature does not exceed 30 °C.
d. Once all ingredients are fully dissolved, adjust the volume to 30 mL with water (MilliQ).
e. Store the prepared solution at 4 °C for up to 4 weeks. In the event of crystallization of any ingredients, remove the solution from the refrigerator, allow it to equilibrate at room temperature for about 1 h, then place it on the magnetic stirrer until the ingredients are fully dissolved. If necessary, heating can be applied (up to 30 °C) to facilitate dissolution.
5. Bradford assay
| BSA (mg/mL) | Volume of BSA (mL) | Volume of H2O (MilliQ) (mL) | Final BSA concentration (mg/mL) |
| 1 | 1 | 0 | 1 |
| 0.75 | 0.25 | 0.75 | |
| 0.5 | 0.5 | 0.5 | |
| 0.25 | 0.75 | 0.25 | |
| 0.2 | 1.8 | 0.1 | |
| 0.1 | 0.75 | 0.25 | 0.075 |
| 0.5 | 0.5 | 0.05 | |
| 0.25 | 0.75 | 0.025 | |
| 0.1 | 0.9 | 0.01 | |
| 0 | 1 | 0 |
Weigh 2 mg of BSA and dissolve it in 2.5 mL of H2O (MilliQ). This solution will be used to generate a standard curve. To prepare additional solutions, dilute the stock solution according to the table. Store all solutions at -20 °C, where they remain stable for up to 6 months.
6. 12% (w/v) polyacrylamide gel
| Reagent | Final concentration | Volume |
|---|---|---|
| 30% acrylamide-bisacrylamide solution 29:1 | 12% | 4 mL |
| 1.5 M Tris-HCl, pH 8.8 | 0.3% | 2.5 mL |
| H2O (MilliQ) | n/a | 3.340 mL |
| 10% SDS | 0.01% | 100 μL |
| 10% APS | 0.05% | 50 μL |
| TEMED | 0.001% | 10 μL |
a. Add the specified quantities of the given solutions to a glass vessel in the order shown in the table.
b. Mix thoroughly by pipetting the mixture several times.
c. Since APS and TEMED initiate polymerization, they should be added last.
d. After all ingredients have been added, mix the gel solution thoroughly, then pour it into the previously prepared glass plates.
e. To limit oxygen exposure, apply a layer of H2O (MilliQ) over the gel.
f. Once the gel has polymerized, it can be used immediately or stored in a humid container at 4 °C for up to 4 days.
7. 1× SDS-PAGE running buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Trizma base | 0.3% | 3.0 g |
| Glycine | 1.44% | 14.4 g |
| SDS | 0.1% | 1.0 g |
| H2O (MilliQ) | n/a | up to 1,000 mL |
a. Weigh the given amount of ingredients and dissolve them in H2O (MilliQ).
b. Place a magnetic stirrer in the bottle and position it on a magnetic stirrer plate.
c. Set the stirring speed to approximately 800 rpm. Optional heating may be applied if necessary.
e. Once all ingredients are completely dissolved, adjust the volume to 1,000 mL with H2O (MilliQ).
f. Store the solution at room temperature for up to 2 months.
8. Gel staining solution
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 100% methanol | 50% | 250 mL |
| 100% acetic acid | 10% | 50 mL |
| Coomassie Brilliant Blue R-250 | 0.06% | 3 g |
| H2O (MilliQ) | n/a | up to 500 mL |
a. Measure the appropriate volumes of methanol and acetic acid as given in the table.
b. Weigh Coomassie Brilliant Blue R-250 and add it to the mixture.
c. After the Coomassie has dissolved, adjust the volume to 500 mL with H2O (MilliQ).
d. Store the solution at room temperature for up to 12 months.
9. Gel destaining solution
| Reagent | Final concentration | Volume |
|---|---|---|
| 100% ethanol | 20% | 200 mL |
| 100% acetic acid | 12% | 120 mL |
| H2O (MilliQ) | n/a | up to 1,000 mL |
a. Measure the appropriate volumes of the given solutions, then adjust the volume to 1,000 mL with H2O (MilliQ).
b. Store the prepared solution at room temperature for up to 6 months.
Laboratory supplies
1. Ceramic mortars and pestles (CHEMLAND, catalog number: 06-J-701)
2. Glass bottles 50 mL (SIMAX, catalog number: S-2070)
3. Glass bottles 100 mL (SIMAX, catalog number: S-2071)
4. Glass bottles 250 mL (SIMAX, catalog number: S-2072)
5. Eppendorf 1.5 mL tubes (BIONOVO, catalog number: B-2277)
6. Eppendorf tubes 2 mL (BIONOVO, catalog number: A-710370)
7. Falcon-type centrifuge tubes with cap Plug-Seal 15 mL (BIONOVO, catalog number: B-0344)
8. Falcon-type centrifuge tubes with cap Plug-Seal 50 mL (BIONOVO, catalog number: B-0345)
9. Pipette tips 10 μL (GenoPlast Biotech S.A., catalog number: GBPT0010-B-N-LB)
10. Pipette tips 200 μL (GenoPlast Biotech S.A., catalog number: GBPT0200-B-Y-LB)
11. Pipette tips 1,000 μL (GenoPlast Biotech S.A., catalog number: GBPT1000-B-N-LB)
12. Weighing vessels 7 mL (Bionovo, catalog number: B-3314)
13. Weighing vessels 100 mL (Bionovo, catalog number: B-3315)
14. Paper towels (Bionovo, catalog number: 2-1416)
15. Semi-micro UV cuvette (Bionovo, catalog number: B-0532)
16. Whatman blotting paper (CL72.1)
Equipment
1. Laboratory scale (RADWAG, model: PS 750.R2)
2. Automatic pipettes 0.5–10 μL, 10–100 μL, 100–1,000 μL (Eppendorf, Adjustable volume pipettes)
3. pH meter (ELMETRON, model: CP-511)
4. Centrifuge (Eppendorf, model: 5415 R)
5. Orbital shaker (SunLab model: SU1020)
6. Spectrophotometer (SHIMADZU, model: UV mini-1240)
7. Mini-PROTEAN® Tetra Cell System (Bio-Rad Laboratories, Inc., catalog number: 1658003)
8. PROTEAN i12 IEF (Bio-Rad Laboratories, Inc., catalog number: 1646001)
Procedure
文章信息
稿件历史记录
提交日期: Jun 6, 2025
接收日期: Aug 26, 2025
在线发布日期: Sep 9, 2025
出版日期: Oct 5, 2025
版权信息
© 2025 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Burchardt, S., Wojtaczka, P., Kućko, A., Ostrowski, M. and Wilmowicz, E. (2025). Advancing 2-DE Techniques: High-Efficiency Protein Extraction From Lupine Roots. Bio-protocol 15(19): e5461. DOI: 10.21769/BioProtoc.5461.
Download Citation in RIS Format
分类
植物科学 > 植物生物化学 > 蛋白质
系统生物学 > 蛋白质组学
生物化学 > 蛋白质 > 电泳
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