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Can anyone help with a simple protocol for chloroplast isolation?
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Aderogbin Francis
Can anyone help with a simple protocol for chloroplast isolation?
For chloroplast isolation first the cell wall is broken mechanically using a blender or homogenizer then filtered to remove the unbroken leaf tissue and the cellular debris . Ref-FEBS Letters Volume 529, Issues 2–3, 9 October 2002, Pages 215-220
For chloroplast isolation first the cell wall is broken mechanically using a blender or homogenizer then filtered to remove the unbroken leaf tissue and the cellular debris . Ref-FEBS Letters Volume 529, Issues 2–3, 9 October 2002, Pages 215-220
Dear Siobhan, I have done 2DE of intact chloroplast isolated fron Rice and Arabidopsis leaves following Napier et al (1996). I tried several protocol but I find this the best with very high effeciency of isolation from leaves. As u have mentioned that you have to isolate intact chloroplast from fruits and mesocarp, u have to modify this protocol accordingly.I also want to make it clear that isolation of intact chloroplast is not a easy task and there is no simple method of chloroplast isolation. You have not mentioned what is the downstream work you have to do with isolated chloroplasts. I am attaching the protocol that may help you in your work
Dear Siobhan, I have done 2DE of intact chloroplast isolated fron Rice and Arabidopsis leaves following Napier et al (1996). I tried several protocol but I find this the best with very high effeciency of isolation from leaves. As u have mentioned that you have to isolate intact chloroplast from fruits and mesocarp, u have to modify this protocol accordingly.I also want to make it clear that isolation of intact chloroplast is not a easy task and there is no simple method of chloroplast isolation. You have not mentioned what is the downstream work you have to do with isolated chloroplasts. I am attaching the protocol that may help you in your work
If you want higher plant chloroplasts that are capable of photosynthesis, or that are at least capable of reducing an electron acceptor, and you need them in quantity, the best source is spinach (Spinacea oleracea) leaves. You can't use just any plant leaves, because many of them contain polyphenol oxidases which will degrade the ability of the chloroplasts to generate the necessary redox potential. Spinach is loaded with good chloroplasts and they isolate easily. You will have to put the leaves in a cold blender with 0.1 M phosphate buffer, pH 7.5 and 0.5 M sucrose to keep them from bursting. After grinding for 30 seconds filter them through 6 layers of cheeze cloth (squeeze them through), and then do a low-speed centrifugation to remove the cell debris. 470 G. discard the pellet. Spin the supernatant at 900G, resuspend the pellet in the grinding buffer and spin them again. That should give you lots of good healthy chloroplasts.
If you want higher plant chloroplasts that are capable of photosynthesis, or that are at least capable of reducing an electron acceptor, and you need them in quantity, the best source is spinach (Spinacea oleracea) leaves. You can't use just any plant leaves, because many of them contain polyphenol oxidases which will degrade the ability of the chloroplasts to generate the necessary redox potential. Spinach is loaded with good chloroplasts and they isolate easily. You will have to put the leaves in a cold blender with 0.1 M phosphate buffer, pH 7.5 and 0.5 M sucrose to keep them from bursting. After grinding for 30 seconds filter them through 6 layers of cheeze cloth (squeeze them through), and then do a low-speed centrifugation to remove the cell debris. 470 G. discard the pellet. Spin the supernatant at 900G, resuspend the pellet in the grinding buffer and spin them again. That should give you lots of good healthy chloroplasts.
you can use phosphate buffer as Philip mentioned above and apply the extract on top of percol gradient in centrifuge tube. After high speed spin (12 000 rpm) chloroplast stay like a band in percol gradient. Look up organelle isolation through percol.
you can use phosphate buffer as Philip mentioned above and apply the extract on top of percol gradient in centrifuge tube. After high speed spin (12 000 rpm) chloroplast stay like a band in percol gradient. Look up organelle isolation through percol.
If you want higher plant chloroplasts that are capable of photosynthesis, or that are at least capable of reducing an electron acceptor, and you need them in quantity, the best source is spinach (Spinacea oleracea) leaves. You can't use just any plant leaves, because many of them contain polyphenol oxidases which will degrade the ability of the chloroplasts to generate the necessary redox potential. Spinach is loaded with good chloroplasts and they isolate easily. You will have to put the leaves in a cold blender with 0.1 M phosphate buffer, pH 7.5 and 0.5 M sucrose to keep them from bursting. After grinding for 30 seconds filter them through 6 layers of cheeze cloth (squeeze them through), and then do a low-speed centrifugation to remove the cell debris. 470 G. discard the pellet. Spin the supernatant at 900G, resuspend the pellet in the grinding buffer and spin them again. That should give you lots of good healthy chloroplasts.
If you want higher plant chloroplasts that are capable of photosynthesis, or that are at least capable of reducing an electron acceptor, and you need them in quantity, the best source is spinach (Spinacea oleracea) leaves. You can't use just any plant leaves, because many of them contain polyphenol oxidases which will degrade the ability of the chloroplasts to generate the necessary redox potential. Spinach is loaded with good chloroplasts and they isolate easily. You will have to put the leaves in a cold blender with 0.1 M phosphate buffer, pH 7.5 and 0.5 M sucrose to keep them from bursting. After grinding for 30 seconds filter them through 6 layers of cheeze cloth (squeeze them through), and then do a low-speed centrifugation to remove the cell debris. 470 G. discard the pellet. Spin the supernatant at 900G, resuspend the pellet in the grinding buffer and spin them again. That should give you lots of good healthy chloroplasts.
Dr Philip Lintilhac , thank you for your input. We have not isolated chloroplast before. Is there a chloroplast isolation kit that you recommend? We are working on a project to sequence chloroplast genome of some Rananculaceae species? Thank you in advance.
Dr Philip Lintilhac , thank you for your input. We have not isolated chloroplast before. Is there a chloroplast isolation kit that you recommend? We are working on a project to sequence chloroplast genome of some Rananculaceae species? Thank you in advance.
For chloroplast isolation first the cell wall is broken mechanically using a blender or homogenizer then filtered to remove the unbroken leaf tissue and the cellular debris .
Ref-FEBS Letters Volume 529, Issues 2–3, 9 October 2002, Pages 215-220
For chloroplast isolation first the cell wall is broken mechanically using a blender or homogenizer then filtered to remove the unbroken leaf tissue and the cellular debris .
Ref-FEBS Letters Volume 529, Issues 2–3, 9 October 2002, Pages 215-220
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Dear Siobhan,
I have done 2DE of intact chloroplast isolated fron Rice and Arabidopsis leaves following Napier et al (1996). I tried several protocol but I find this the best with very high effeciency of isolation from leaves. As u have mentioned that you have to isolate intact chloroplast from fruits and mesocarp, u have to modify this protocol accordingly.I also want to make it clear that isolation of intact chloroplast is not a easy task and there is no simple method of chloroplast isolation. You have not mentioned what is the downstream work you have to do with isolated chloroplasts. I am attaching the protocol that may help you in your work
Dear Siobhan,
I have done 2DE of intact chloroplast isolated fron Rice and Arabidopsis leaves following Napier et al (1996). I tried several protocol but I find this the best with very high effeciency of isolation from leaves. As u have mentioned that you have to isolate intact chloroplast from fruits and mesocarp, u have to modify this protocol accordingly.I also want to make it clear that isolation of intact chloroplast is not a easy task and there is no simple method of chloroplast isolation. You have not mentioned what is the downstream work you have to do with isolated chloroplasts. I am attaching the protocol that may help you in your work
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Hi Slobhan,
Do you want to isolate intact chloroplasts to perform further research with them?
Frank
Hi Slobhan,
Do you want to isolate intact chloroplasts to perform further research with them?
Frank
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If you want higher plant chloroplasts that are capable of photosynthesis, or that are at least capable of reducing an electron acceptor, and you need them in quantity, the best source is spinach (Spinacea oleracea) leaves. You can't use just any plant leaves, because many of them contain polyphenol oxidases which will degrade the ability of the chloroplasts to generate the necessary redox potential. Spinach is loaded with good chloroplasts and they isolate easily. You will have to put the leaves in a cold blender with 0.1 M phosphate buffer, pH 7.5 and 0.5 M sucrose to keep them from bursting. After grinding for 30 seconds filter them through 6 layers of cheeze cloth (squeeze them through), and then do a low-speed centrifugation to remove the cell debris. 470 G. discard the pellet. Spin the supernatant at 900G, resuspend the pellet in the grinding buffer and spin them again. That should give you lots of good healthy chloroplasts.
If you want higher plant chloroplasts that are capable of photosynthesis, or that are at least capable of reducing an electron acceptor, and you need them in quantity, the best source is spinach (Spinacea oleracea) leaves. You can't use just any plant leaves, because many of them contain polyphenol oxidases which will degrade the ability of the chloroplasts to generate the necessary redox potential. Spinach is loaded with good chloroplasts and they isolate easily. You will have to put the leaves in a cold blender with 0.1 M phosphate buffer, pH 7.5 and 0.5 M sucrose to keep them from bursting. After grinding for 30 seconds filter them through 6 layers of cheeze cloth (squeeze them through), and then do a low-speed centrifugation to remove the cell debris. 470 G. discard the pellet. Spin the supernatant at 900G, resuspend the pellet in the grinding buffer and spin them again. That should give you lots of good healthy chloroplasts.
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You can follow this protocol: https://microbenotes.com/protocol-of-chloroplast-isolation/
You can follow this protocol: https://microbenotes.com/protocol-of-chloroplast-isolation/
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you can use phosphate buffer as Philip mentioned above and apply the extract on top of percol gradient in centrifuge tube. After high speed spin (12 000 rpm) chloroplast stay like a band in percol gradient. Look up organelle isolation through percol.
you can use phosphate buffer as Philip mentioned above and apply the extract on top of percol gradient in centrifuge tube. After high speed spin (12 000 rpm) chloroplast stay like a band in percol gradient. Look up organelle isolation through percol.
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PEG method is a siple method to isolate the protoplasts.....
PEG method is a siple method to isolate the protoplasts.....
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If you want higher plant chloroplasts that are capable of photosynthesis, or that are at least capable of reducing an electron acceptor, and you need them in quantity, the best source is spinach (Spinacea oleracea) leaves. You can't use just any plant leaves, because many of them contain polyphenol oxidases which will degrade the ability of the chloroplasts to generate the necessary redox potential. Spinach is loaded with good chloroplasts and they isolate easily. You will have to put the leaves in a cold blender with 0.1 M phosphate buffer, pH 7.5 and 0.5 M sucrose to keep them from bursting. After grinding for 30 seconds filter them through 6 layers of cheeze cloth (squeeze them through), and then do a low-speed centrifugation to remove the cell debris. 470 G. discard the pellet. Spin the supernatant at 900G, resuspend the pellet in the grinding buffer and spin them again. That should give you lots of good healthy chloroplasts.
If you want higher plant chloroplasts that are capable of photosynthesis, or that are at least capable of reducing an electron acceptor, and you need them in quantity, the best source is spinach (Spinacea oleracea) leaves. You can't use just any plant leaves, because many of them contain polyphenol oxidases which will degrade the ability of the chloroplasts to generate the necessary redox potential. Spinach is loaded with good chloroplasts and they isolate easily. You will have to put the leaves in a cold blender with 0.1 M phosphate buffer, pH 7.5 and 0.5 M sucrose to keep them from bursting. After grinding for 30 seconds filter them through 6 layers of cheeze cloth (squeeze them through), and then do a low-speed centrifugation to remove the cell debris. 470 G. discard the pellet. Spin the supernatant at 900G, resuspend the pellet in the grinding buffer and spin them again. That should give you lots of good healthy chloroplasts.
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Mason et al. 2006 - nature methods: A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii
Mason et al. 2006 - nature methods: A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii
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To isolate a high number of chloroplasts (over 90%) efficiently, use the
Minute™ Chloroplast Isolation Kit. It only needs 50-200 mg plant tissue to yield a high amount of intact chloroplasts in just 5 minutes. https://inventbiotech.com/products/minute-chloroplast-isolation-kit?_pos=1&_sid=b4c76f0e0&_ss=r
To isolate a high number of chloroplasts (over 90%) efficiently, use the
Minute™ Chloroplast Isolation Kit. It only needs 50-200 mg plant tissue to yield a high amount of intact chloroplasts in just 5 minutes. https://inventbiotech.com/products/minute-chloroplast-isolation-kit?_pos=1&_sid=b4c76f0e0&_ss=r
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Dr Philip Lintilhac , thank you for your input. We have not isolated chloroplast before. Is there a chloroplast isolation kit that you recommend? We are working on a project to sequence chloroplast genome of some Rananculaceae species? Thank you in advance.
Dr Philip Lintilhac , thank you for your input. We have not isolated chloroplast before. Is there a chloroplast isolation kit that you recommend? We are working on a project to sequence chloroplast genome of some Rananculaceae species? Thank you in advance.
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