This method can help answer key questions about the composition and dynamic organization of the photosynthetic protein complexes in plant thylakoid membrane. The main advantage of this technique is that it allows the analysis of functional interactions between native protein complexes and elucidates the plasticity of the protein complex organization in varying environmental conditions. Set up the gel caster eight-centimeter by 10-centimeter plates according to manufacturer's instructions using 0.75-millimeter spacers.
Place a gradient mixer on a stir plate and connect it with the peristaltic pump by tubing. Attach a syringe needle to the other end of the tubing and place the needle between the glass and aluminum plate. Place a magnetic stirrer in the heavy chamber.
Prepare 3.5%and 12.5%acrylamide solutions in 15-milliliter conical centrifuge tubes as detailed in the text protocol to use for the separation gel gradient. To prevent untimely polymerization keep the centrifuge tubes on ice while preparing the solutions. Add 5%APS and TEMED right before pippetting the solutions to the gradient mixer.
Pipette the 12.5%solutions to the heavy chamber. Remove air bubbles from the channel connecting the light and heavy chamber by opening the valve connecting the two chambers allowing solution to enter to the light chamber. Close the valve and pipette the traces of solution back to the heavy chamber.
Finally, pipette the 3.5%solution to the light chamber. Switch on the magnetic stirrer. Open the valves, and switch on the peristaltic pump.
Allow the gel solutions to flow between the glass and aluminum plate at a flow rate of roughly 0.5 milliliters per minute. The needle must be above the liquid at all times. When the heavy and light chambers have emptied fill them with ultrapure water.
And allow water to gently overlay the gel surface. Gel polymerization takes about one to two hours at room temperature. Prepare the 3%acrylamide solution for the stacking gel at room temperature.
Pipette the stacking gel on top of the polymerized separation gel and place a sample gel comb between the glass and aluminum plate avoiding air bubbles. After allowing the gel to polymerize for 30 to 60 minutes at room temperature, remove the comb gently under ultrapure water. At this point the gel can be stored at positive four degrees Celsius in moist conditions for a few days.
Dilute isolated thylakoids with ice-cold 25BTH20G buffer to a final chlorophyll concentration of one milligram per milliliter. Prepare two tubes for each thylakoid sample to perform both beta-DM and digitonin solubilization. Now add an equal volume of detergent buffer.
Mix the detergent and thylakoid sample gently with a pipette and avoid making air bubbles. Solubilize the thylakoids for two minutes on ice for the beta-DM solubilization. Or 10 minutes at room temperature with continuous gentle mixing on a rocker or shaker for the digitonin solubilization.
Following incubation, remove the insolubilized material by centrifugation at 18, 000 g for 20 minutes at positive four degrees Celsius. Transfer the supernatant to a new 1.5-milliliter tube and add 1/10 volume of Coomassie brilliant blue buffer to the sample. Set up the gel in a vertical electrophoresis system.
Fill the upper buffer chamber with blue cathode buffer and pour anode buffer to the lower buffer chamber. Load the thylakoid samples into the wells. Start the electrophoresis and gradually increase the voltage as listed in the text protocol.
Run the gel at positive four degrees Celsius either using a cold room or adjusting the gel temperature with a cooling system. Change the blue cathode buffer to a clear cathode buffer when the sample front has migrated about 1/3 of the gel. After the electrophoretic run scan the gel with a photo scanner for image archiving.
To perform 2-SDS PAGE assemble the vertical electrophoresis system using one-millimeter spacers. Prepare a standard SDS gel with a 2-D comb as described in the text protocol. Cut the lane from the BN gel and place it in a five-milliliter tube.
Then add two milliliters of Laemmli buffer and incubate the strip for 45 minutes with gentle shaking at room temperature. Place the lane with the help of a spacer on top of the gel avoiding air bubbles. Pipette five microliters of molecular weight marker on a narrow piece of filter paper and place the paper in the standard well.
To seal the BN gel strip and the marker paper pour a 0.5%agarose in running buffer on top of the gel strip and allow it to solidify. Perform electrophoresis according to standard protocols. After the electrophoretic run visualize the proteins with SUPRO Ruby stain or silver staining.
Representative results of thylakoid protein complex patterns of beta-DM and digitonin-solubilized samples are shown here. The small complexes migrate faster during the electrophoretic run and are therefore in the lower part of the gel. Whereas the large complexes are in the upper part of the gel.
Digitonin generally preserves protein complexes in larger assemblies, while beta DM interferes with labile protein-protein interactions between the complexes. The subunit composition of protein complexes of beta-DM and digitonin-solubilized thylakoids are presented here. The subunits of each complex are found from the vertical line below the aligned complex.
In principle, each spot in the 2-D gel represents an individual protein. But in practice, several proteins of same molecular mass may be present in a single spot. The identification of the spots is based on mass spectrometry analysis.
After applying this procedure one can continue with other methods like electron microscopy and single particle analysis of the protein complexes eluted from the blue native gel in order to get structural insights into the protein complexes. To identify unknown protein subunits the individual protein spots from two-dimensional SDS PAGE can be analyzed with mass spec. Don't forget that working with acrylamide and digitonin can be extremely harmful and precautions such as wearing protective gloves should always be taken while performing this procedure.
