The main advantage of this technique is that it allows distinguishing proteins of similar activities that have different roles, depending on the localization in envelope, stroma, or thylakoid membranes. Generally, individuals new to this method will struggle, because they will use plants that are too old, blend the leaf for too long, or won't be careful enough when re-suspending the pellets of intact, but fragile, chloroplast. Visual demonstration of this method is critical, as the loading or recovering of fraction from percoll or in sucrose gradient steps are difficult to learn.
Because of the risk of blending of the gradients and thus of cross-contaminating the various chloroplast sub-fractions. Demonstrating the procedure will be by Imen Bouchnak, a PhD student from our laboratory and Lucas Moyet, an indoor engineer. To begin, grow arabidopsis plants for five weeks with a 12 hour light cycle at 23 degrees Celsius in the day and 18 degrees Celsius in the night, with a light intensity of 150 micromoler per square meter per second.
The next day, pre-weigh a five liter beaker and then place it on ice. Harvest the arabidopsis leaves, making sure to avoid picking any soil. Re-weigh the beaker and record the tissue weight.
Transfer the harvested leaves to a cold room. Place the leaves in a blender containing two liters of grinding buffer and homogenize them by blending at high speed three times for a duration of two seconds each time. Place four layers of muslin and one layer of nylon blue tex into a strainer and this to filter the homogenate.
Gently squeeze the blended leaves inside the muslin and blue tex to extract all of the liquid. Recover the remaining tissue in the blender cup and repeat the homogenization process using fresh grinding buffer. Use four to five layers of new muslin to filter this new homogenate in a new beaker as previously described.
Distribute the crude cell extract equally into six 500 milliliter bottles. Place the bottles on ice. Centrifuge the chilled bottles at 2070 G and four degrees Celsius for two minutes.
After this, gently discard the supernatant. Use a water pump to aspirate any remaining supernatant and keep the pellets, which contain concentrated crude chloroplast, on ice. Using a 10 milliliter pipette, add three milliliters of washing medium to each bottle, making sure not to use fine pipette tips to avoid breaking the chloroplasts.
Then, use a paint brush or curved plastic spatula to gently re-suspend the pellets. Using a 10 milliliter pipette, collect the re-suspended chloroplasts in a single tube. Gently invert the tube to obtain a homogenous chloroplast suspension.
To begin, use a 10 millileter pipette to slowly load six milliliters of the chloroplast suspension on top of each of the six prepared percoll gradients. Using a swinging bucket rotor, centrifuge the gradients at 13, 300 G and four degrees Celsius for 10 minutes. Use a water pump to aspirate the upper phase, which contains broken chloroplasts and intact mitochondria.
Then, use a 10 milliliter pipette to retrieve intact chloroplasts present in the lower phase, being careful not to aspirate the nuclei and cell debris found at the bottom of the tube along with the intact chloroplasts. Dilute the intact chloroplast suspension three to four fold with washing medium. Keep approximately 10 milliliters of an aliquot of intact chloroplast fraction for further analysis by SDS-Page and Western blotting.
Centrifuge at 2070 G and four degrees Celsius for two minutes. After this, gently discard the supernatant. Use a water pump to aspirate any remaining supernatant and keep the pellet, which contains concentrated crude chloroplasts, on ice.
To lyse the purified intact chloroplasts, re-suspend the pellet in hypotonic medium that contains protease inhibitors. Transfer the re-suspended chloroplasts to a 10 milliliter falcon tube. Prepare the sucrose gradient as outlined in the text protocol.
Using a peristaltic pump, slowly load three milliliters of the lysed chloroplasts on top of each of the pre-formed sucrose gradients. Use hypotonic medium buffer to balance pairs of tubes, and then ultra-centrifuge the gradient at 70, 000 G and four degrees Celsius for one hour. Take an aliquot of the soluble stromal proteins to be used in the determination of the protein concentration.
Next, use a water pump to aspirate the remaining upper phase of the gradient up to the yellow band. Using a pipette, retrieve the yellow band, which is the envelope. Pull the envelopes into one tube.
Then, remove the remaining phase of the gradient up to the thylakoid pellet. Re-suspend the thylakoid pellets in membrane washing buffer with protease inhibitors. Dilute the thylakoid suspension and envelope three to four fold with washing medium, adjusting the final volume to 10 milliliters.
Balance pairs of tubes with membrane washing buffer and ultra-centrifuge them at 110, 000 G and four degrees Celsius for one hour. After this, use a water pump to carefully aspirate the supernatant. Add approximately 100 microliters of membrane washing buffer to the envelope pellet.
Then, aspirate the supernatant from the thylakoid tube. Re-suspend the thylakoid pellets in three milliliters of membrane washing buffer. Store the three purified sub-compartments in liquid nitrogen for use in further experiments.
After the membrane fractions are recovered, washed, and concentrated, SDS-Page is used to quantify the proteins and analyze the composition of all four fractions. The most abundant protein from the stroma is RBCL, and representative results show the expected result, that very little of the large subunit of this protein is contained the thylakoid and envelope membrane fractions. The light harvesting complex proteins are abundant thylakoid components that should barely contaminate the envelope membranes.
Lastly, the phosphate triose phosphate translocator is only visible in the purified envelope fraction, due to its strong enrichment in the envelope fraction when compared to the whole chloroplast extracts. Cross-contamination of the three sub-compartments, the soluble ketol-acid reductoisomerase from the stroma, the chloroplast envelope copper ATPase, and the light harvesting complex proteins from the thylakoid membranes can be quantified by using both immunoblotting and mass spectrometry analysis. While the stroma is not usually contaminated by envelope or thylakoid fractions, the purified envelope fractions contain 3%thylakoid proteins and up to 10%of proteins from the stroma.
The thylakoids are poorly contaminated by proteins from the stroma, but contain up to 3%of envelope membrane proteins. Chloroplasts perform many crucial functions such as assimilation of carbon, sulfur, and nitrogen, as well as synthesis of essential metabolites. In order to decipher new regulatory mechanisms that control the chloroplast physiology, defining the surplasty localization of chloroplast proteins is critical to super-targeted studies.
Chloroplasts contain several sub-compartments. This method can help answer key questions in the chloroplast field, such as where specific chloroplast protein is located within the organelle. While attempting this procedure, it's important to remember to conduct all of the chloroplast isolation steps at four degrees to remit protea resist into purified fractions.
Following this procedure, other methods like lascare proteomic approaches can be performed in order to answer additional questions like the composition and dynamics of the proteome of the values plastid sub-compartments. After its development, this technique paved the way for researchers in the field of plant physiology to explore many different aspects of the chloroplast biogenesis and function using targeted analysis of candidate proteins identified within plastid sub-compartments. Don't forget that working with volume blender, liquid nitrogen, on specific compounds like protease inhibitors, requires special care.
Precautions such as wearing gloves, lab coat, and safety glasses should always be taken while performing this procedure.
