Identification and analysis of plant protein complexes by blue-green nondenaturing gel electrophoresis experiments
High-resolution protein separation is an essential requirement for proteomics studies, and protein separation relies heavily on 2D IEF/SDS-PAGE; unfortunately, this technique is poor at separating hydrophobic proteins and cannot be used to study nondenatured proteins in their natural state. The source of this experiment is the "Guide to Plant Proteomics Experiments" [France] H. Tillmant, M. Zivi, C. Damerweil, V. Mitschine, eds.
Operation method
Identification and analysis of plant protein complexes by blue-green non-denaturing gel electrophoresis
Materials and Instruments
Dodecyl β-D-maltoside booster solution TritonX-100 booster solution Digitalis fucoidan booster solution Khao Maas Brilliant Blue Staining solution Acrylamide solution BN Gel buffer Move 3.1 BN-PAGE Sample Preparation For more product details, please visit Aladdin Scientific website.
All sample preparation steps must be performed at 4°C. Before starting sample preparation (see Sections 26.3.1 1 and 26. 3.1 2), prepare a BN gel (see 26. 3. 2).
1. Membrane Components
( 1 ) Prepare the membrane fractions to be studied, such as cytoplasmic, vesicular, or mitochondrial membranes, according to standard method steps. Alternatively, use intact organelles, such as plastids, mitochondria, or peroxisomes, as the initial experimental material for BN-PAGE.
( 2 ) Determine the protein concentration, e.g., using the Lowry method.
( 3 ) Adjust the protein concentration to 10 μg/μl.
( 4 ) Centrifuge 100 μl sample at 15000 g for 10 min to precipitate membrane or organelle fractions.
( 5 ) Suspend the precipitate with dodecyl β-D-maltoside, Triton X-100, or digitalis saponin-enhanced solution (see Note 1).
( 6 ) Place the suspension on ice for 15 min.
( 7 ) Centrifuge at 20,000 g for 20 min to remove the inadmissible material.
( 8 ) Add 5 μL of komas Brilliant Blue Staining Solution.
( 9 ) Spike 50~100 μl of supernatant (equivalent to 0.5~1.0 mg of protein) directly onto the BN gel (the amount of protein is quantified as the amount of protein that can be stained with Khomas Brilliant Blue, and the amount of protein can be reduced to 1/10 if stained with silver).
2. Water soluble fractions
( 1 ) Prepare the water-soluble proteins to be studied according to standard methods, using a buffer that does not contain high salt concentrations or ionic detergents. The protein concentration should be adjusted to 10 μg/μl.
( 2 ) Add 2 μl of the Khomas Brilliant Blue Staining Solution to 100 μl of the sample. 
( 3 ) Centrifuge at 20,000 g for 20 min to remove insoluble material.
( 4 ) Spike 50-100 μl of supernatant (equivalent to 0.5-1.0 mg of protein) directly onto the BN gel (if silver staining is used, reduce the amount of protein sample spiked, see 26. 3.1 Section 1, step (a)).
3.2 BN-PAGE
The best resolution is achieved with BN gels if the electrophoretic separation distance is greater than 12 cm. The method described below was performed on a Bio-Rad Protein II electrophoresis machine (Bio-Rad, Richmond, CA; gel size: 0.15 cm X 16 cm X 20 cm), but other electrophoresis machines such as the Hoefer SE-400 or SE-600 electrophoresis machines (GE Healthcare, Munich, Germany) are also available. Munich, Germany), are also suitable for BN-PAGE electrophoresis. Since the molecular mass of protein complexes can range from 50 kDa to several thousand kDa, gradient gels are sometimes used for separation (see Note 2).
( 1 ) Prepare a 4.5% separation gel solution by mixing 1.8 ml of acrylamide solution, 3.3 ml of BN gel buffer, and 14.9 ml of double-distilled water.
( 2 ) Prepare a 16% Separation Gel Solution by mixing 6.7 ml Acrylamide Solution, 3.3 ml BN Gel Buffer, 6.0 ml Double-Distilled Water, and 4 ml Glycerin.
( 3 ) Fill each of these two solutions into a gradient generator to which a hose and needle are attached and connected to the space between the two glass plates where the gel is made, so that the gel can be filled from the top (16% Separation Gel Solution is filled between the two glass plates first) or from the bottom (4.5% Separation Gel Solution is filled between the two glass plates first).
( 4 ) Add TEMED and APS to both gel solutions ( 90 μl 10% APS/9 μl TEMED to 4.5% Separation Gel Solution; 65 μl APS/6.5 μl TEMED to 16% Separation Gel Solution).
( 5 ) Fill the gradient gel, leave some space at the top for the concentrated gel, seal the gel solution with double-distilled water, and the gel will polymerize within 60 min.
( 6 ) Pour off the double-distilled water.
( 7 ) Prepare a concentrated gel solution by mixing together 1.2 ml of acrylamide solution, 2.5 ml of BN gel buffer, and 11.3 ml of double-distilled water.
( 8 ) Add 65 μl of APS and 6.5 μl of TEMED to the gel concentrate solution and pour it into the space around the insertion comb, the gel concentrate will polymerize within 30 min.
( 9 ) Dilute the concentrated reservoir of the corresponding electrophoresis buffer to prepare 1X anodic and cathodic electrophoresis buffers, respectively.
( 10 ) When the gel concentrate has been polymerized, carefully remove the insert comb.
( 11 ) Add the anodic and cathodic electrophoresis buffers to the upper and lower tanks of the electrophoresis apparatus respectively, and cool the electrophoresis apparatus to 4 °C.
( 12 ) Spike the protein samples pretreated with Kaomas Brilliant Blue into the gel recesses.
( 13 ) Connect the electrophoresis apparatus to a stabilized power supply and electrophoresis at a constant voltage of 100 V for 45 min at the beginning, followed by electrophoresis at a constant current of 15 A for 8 h. To electrophoresis at 4°C, the bands on the BN gel can be seen during the electrophoresis process.
3.3 SDS-PAGE of the second phase
Protein complexes isolated by 1D BN-PAGE can be stained with Cauloblue or silver, assayed for activity by in-gel enzymology, or transferred to a blotting membrane for further analysis (see Note 3 and Note 4). Gel electrophoresis with a second phase in the presence of SDS separates the subunit components of protein complexes, and all published SDS-PAGE assays can be combined with BN-PAGE, such as the electrophoretic system published by Laemmli [24] . In general, however, the Tricine-SDS-PAGE system developed by Schagger and von Jagow [ 25 ] provides the highest resolution. The method described below was performed on a Bio-Rad Protein II electrophoresis apparatus (Bio-Rad, Richmond, C A; gel size: 0.15 cm X 16 cm X 20 cm), and electrophoresis apparatus manufactured by other companies are equally suitable.
( 1 ) Cut a strip of BN gel and soak it in denaturing solution for 30 min at room temperature.
( 2 ) Rinse the strip with double-distilled water for 30-60 s (this step is important because β-mercaptoethanol inhibits the polymerization of acrylamide).
( 3 ) Place the adhesive strip on the glass plate of the electrophoresis apparatus in the comb position of the normal adhesive comb.
( 4 ) Assemble the gel electrophoresis apparatus with another 1 mm rubber seal, a second glass plate, and clamps. Pour everything you need into the gel dispenser (reduce the thickness of the second gel to 1.0 mm compared to the thickness of the gel strip in the first direction (1.5 mm) to avoid the second gel strip from sliding down along the vertical).
( 5 ) Prepare a 16% separating gel solution by mixing together 10 ml of acrylamide solution, 10 ml of SDS gel buffer, 10 ml of double-distilled water, 100 μl of APS, and 10 μl of TEMED, and pour it into the space below the BN tape between the two glass plates of the electrophoresis apparatus, leaving space for the sample gel solution embedded in the BN tape. The gel surface was sealed with the sealing solution and the gel was polymerized within approximately 60 min.
( 6 ) Prepare a 10% sample gel solution by mixing together 4.1 ml of acrylamide solution, 6.7 ml of BN gel buffer, 2 ml of glycerin, 200 μl of SDS solution, 6.8 ml of double-distilled water, 160 μl of APS and 16 μl of TEMED.
( 7 ) Pour off the sealer solution, pour in the sample gel to embed the BN tape, and tilt the gel rack to release the air under the BN tape.
( 8 ) Add SDS anodic and cathodic electrophoresis buffers to the upper and lower tanks of the electrophoresis apparatus, respectively.
( 9 ) Connect the electrophoresis apparatus to a regulated power supply and electrophoresis at 30 mA overnight.
As an alternative system, second direction electrophoresis can be performed under natural conditions (see Note 7).
3.4 Staining
Proteins isolated on 2D BN gels can be stained using all standard protein staining methods, such as Caulforan, silver, fluorescent dyes, etc. If the proteins are then identified by mass spectrometry, it is recommended to use Caulforan. If the proteins are then identified by mass spectrometry, it is recommended to use Caumas Blue staining, as described in the following experimental guide based on gel Caumas Blue staining developed by Nenhoff et al.
( 1 ) Fix the gel in a fixative for 1 h. The gel should be treated with a fixative.
( 2 ) Mix 80 ml of freshly prepared staining solution and 20 ml of ethanol solution.
( 3 ) Soak the gel in the above solution overnight.
( 4 ) Rinse the gel with double-distilled water to remove the color, changing the water halfway until the background is clear (if the background color cannot be removed with water, 20% ethanol solution can be used to remove the color).