The overall goal of this procedure is to comparatively analyze the composition of MultiPro complexes in thal cord membranes under different conditions. This is accomplished by first isolating thal membranes from lamona cells exposed to anaerobic conditions. Next, the th cord membranes are solubilized and fractionated on a sucrose density gradient.
Then equal volumes of the desired fractions are mixed and separated on SDS page. Finally, the kumasi stained bands are digested with trypsin. Ultimately, samples are analyzed by quantitative mass spectrometry to observe changes in protein abundance between the investigated fractions.
The main advantage of this technique over existing methods like quantitative proteomic studies comparing a defined amount of protein is that in our approach, equal volumes of fractionated samples are combined and analyzed. This allows studying the migration behavior of proteins within the gradient, and moreover, to analyze the composition of different non-protein complexes in the third group membrane. With respect to the applied strains After culturing chlamydomonas rein hardy strains, according to the text protocol, use stock solutions of two molar sucrose, 10%beta DM in water, and 0.5 molar trice pH 8.0.
To prepare the following solutions for sucrose density gradients to set up the gradients using 14 by 89 millimeter centrifuge tubes begin by slowly pouring one milliliter of 1.3 molar solution, followed by one milliliter of 1.0 molar solution. Then pour two milliliters each of the 0.85, 0.7 0.65, and finally, the 0.4 molar solutions. Store the gradients overnight in the cold room to induce anaerobic conditions.
In the chlamydomonas cultures, insert a glass pipette into culturing flasks and bubble with Argonne for four hours with continuous stirring and illumination for isolating thal cord membranes. After the incubation, begin by pelleting the cells for five minutes at 2, 500 times gravity and four degrees Celsius. Then resus suspend the cell pellets in 30 milliliters of H one buffer.
Repeat the centrifugation and again, resus. Suspend the cells in H one buffer to break the cells, passage them through a nebulizer with a nitrogen pressure of 1, 500 Pascals. Make sure that the distance between the ceramic ball and the metal is small enough for cells to break.
Then spin down the cells for seven minutes at 2, 500 times gravity and four degrees Celsius. The supernatant should be light green in color. If the cell breakage was successful, resus suspend the cells in 50 milliliters of H two buffer and pellet them for 10 minutes at 32, 800 times gravity and four degrees Celsius.
Then after Resus suspending the cells in 10 milliliters of H three buffer, use a potter to homogenize the resuspended palate. Next to prepare thal cord sucrose density gradients. Begin with 12 milliliters of cells in H three buffer.
Then slowly pour a layer of 12 milliliters of H four buffer and finish with 12 milliliters of H five buffer. Use H five to balance the rotor and centrifuge the gradients for one hour at 70, 700 times gravity. Remove the TH bands from the gradients and use an appropriate volume of H six buffer to dilute them.
Spin down cells at 37, 900 times gravity for 20 minutes at four degrees Celsius. If the pellet is not tight, add more H six buffer to the centrifugation step. Then resus.
Suspend the thal cords in a small volume of H six buffer to load a photo system sucrose density gradient. First, calculate the amounts of thal cords, beta DM and H six buffer needed for each gradient. Following these guidelines, each gradient will contain 0.8 milligrams per milliliter of chlorophyll in 0.9%beta DM and H six Buffer will bring the final volume up to 700 microliters.
To solubilize the thys, prepare separate Eloqua with Thylakoids beta DM and H six buffer for each gradient. Incubate the samples on ice for 20 minutes and invert every few minutes to mix. After centrifuging the samples at 14, 000 times gravity for 10 minutes and four degrees Celsius.
The pellet should be small and whitish, and the supernatant will be dark green. Load the supernatant onto the gradients balance with H six buffer and ultra centrifuge at 134, 470 times gravity for 14 hours and four degrees Celsius. After the spin, take pictures of the gradients then to fractionate.
Use a needle to puncture a hole at the bottom of the tube and collect fractions in 500 microliter tubes. Or a 96 well plate process. The samples for SDS page in gel digestion and mass spectrometry according to the text protocol as shown here, using the results from immuno blood analysis fraction six from wild type and fraction 13 from delta PS one.
Peak fractions of A and R one were chosen for analysis of cyclic electron flow super complex components in this figure. Relative protein ratios depicted as wild type over delta PS one 14 N over 15 N for several PS one core and light harvesting proteins of PS one as well as for proteins assigned with the cyclical electron flow super complex are shown the differences in abundance of a NR one and CAS in fraction six and 13 of wild type and delta PS one suggest that they are novel components of this complex shown here are the results for the quantitative comparison of this cyclical electron flow super complex between the wild type and a PG L one knockout strain. Interestingly, HCF 1 36, cytochrome B six, cytochrome B six F subunit four and PET C seem to be enriched in the wild type while FTSH two, cytochrome F and PET O seem to be enriched in PG L one.
After watching this video, you will have a good understanding of how to compare the composition of MultiPro complexes in di liquid membranes under different conditions. Keep in mind that for the successful preparation of th liquid membranes and isolation of the cyclic electron flow, super complex cell disruption, pottering of cells and ization of th liquid membranes are the critical steps. Furthermore, the sucrose density gradients need to be centrifuge for at least 12 hours to achieve full separation of photosynthetic complexes.
