This method can help answer key questions in the chloroplast field, for instance, the composition of the chloroplast protein import machinery. It is essential for greening and photosynthesis and hence for food production on the planet. The molecular machine consists of two separate complexes in the outer and inner member of the chloroplast called TOC and TIC, but the composition is not fully known.
The main advantage of this technique is that the purification of Arabidopsis TOC/TIC complex can be accomplished in a single step purification method, TAP-TOC purification. It is a specific and efficient method. Generally, researchers new to this method may struggle because they're not familiar with the isolation of chloroplast membrane fractions, detergent, solubilization, or affinity purification.
After preparing the Arabidopsis plants, grind 10 grams of three week old seedlings in liquid nitrogen, using a cold mortar and pestle with sand. Add 20 milliliters of cold grinding buffer to the ground tissues. Mix well and let the mixture thaw on ice.
Next, soak two layers of quick filtration material with grinding buffer. Filter the homogenate through the two layers of filtration material into a 50 milliliter conical centrifuge tube. Centrifuge the filtrate at 1, 500 times g in four degrees Celsius for 10 minutes and transfer the supernatant to a fresh, pre-chilled 50 milliliter conical centrifuge tube.
Repeat the centrifugation once more at the same conditions. Transfer the supernatant to a cold 38.50 milliliter ultracentrifuge tube and top it up to 35 milliliters with cold grinding buffer. Use an ultracentrifuge to centrifuge the sample at 100, 000 times g and four degrees Celsius for one hour.
Using a glass Teflon homogenizer, resuspend the green pellet in grinding buffer. Centrifuge at 100, 000 times g and four degrees Celsius for one hour and discard the supernatant. Use the glass Teflon homogenizer to resuspend the green pellet in 18.75 milliliters of one x grinding buffer.
Then add another 9.375 milliliters of one x grinding buffer. Add 9.375 milliliters of four x solubilization solution to bring the final volume to 37.5 milliliters and incubate on a rotating shaker at four degrees Celsius for 30 minutes. After this, use an ultracentrifuge to centrifuge the sample at 100, 000 times g and four degrees Celsius for one hour.
Transfer the supernatant containing the solubilized membranes to a fresh 50 milliliter conical centrifuge tube. After preparing the IGG agarose resin, transfer the previously washed IGG agarose resin to the 50 milliliter conical tube containing the 37.5 milliliters of solubilized membranes. Incubate over night on a rotating shaker at four degrees Celsius.
The next day, sediment the IGG agarose resin at 100 times g and four degrees Celsius for five minutes. Use a pipette to remove the supernatant. Was the IGG agarose resin in 37.5 milliliters of buffer A and incubate on a rotating shaker at room temperature for 10 minutes.
Continue sedimenting and washing the resin as outlined in the text protocol. After this, transfer the IGG agarose resin to a 500 microliter spin column with a 35 micron filter. Wash the beads twice with 300 microliters of TEV elution buffer for equilibration.
Next, add 300 microliters of TEV elution buffer contain 10 microliters of TEV protease. Incubate the spin column with beads in a Thermomixer at 16 degrees Celsius and 350 rpm for two hours. Then, open the spin column and place it in a new, clean, 1.5 milliliter tube.
Spin this tube at 100 times g and four degrees Celsius for five minutes to collect the eluate. In this study a TAP tagged chloroplast envelope protein complex from transgenic A.thaliana plants is purified. Immunoblot analysis confirms that isolate TAP-TOC159 interacts with TOC75 and TOC33 of the TOC complex.
The chloroplast outer membrane kinase, KOC1, which is known to phosphorylate TOC159 is also co-isolated. The presence of TIC110 reveals that the isolated TAP-TOC159 complex also contains the components of the TIC complex. The FBN1A antibody did not recognize the TAP-TOC159 complex, indicating the absence of contaminations.
In the negative control, the immunoprecipitated NTAP protein did not co-isolate with any of the TOC, TIC proteins and confirms the specificity of the TAP-TOC159 purification. Though this method can provide insight into chloroplast protein import, it can potentially be applied to any other complex in the Arabidopsis thaliana bottle system. Following this procedure, other methods can be applied such as western blotting or mass spectrometry to either demonstrate the presence of known components or to identify new components of the TOC and TIC complexes.
This technique paved the way for research in the chloroplast protein import field to explore the function of new components of the TOC and TIC complexes.
