What is the function of sucrose in lysis buffers for isolation of intact nuclei? I observed that nuclei pellets resuspend easier in buffers without sucrose, and have a higher tendency to clump when sucrose (0.3M) is present. Clumping does not seem a good thing to me. So is there any other function of sucrose apart from being a density gradient medium for subsequent purification?
Most buffers would contain 0.25-0.3M sucrose. Why?
-Apologize for lengthiness-
Ok, dear Lech, Good morning.
Would you mind to provide the protocol (and/or source or Reference of the protocol) or at least a more detailed description of the lysis buffer you are using ??
Concerning the other matter:
i) the criticized WIKI-link:
Read in the WIKI-compilation:
ADDITIVES/OTHER:
Other additives include metal ions, sugar like glucose *) , glycerol, reducing agents like dithiothreitol (DTT).
*) and "sugar" might be also Saccharose=Sucrose: cf: reference [4]:
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/
find ADDITIVES (CTRL-F, insert | additives | Return ) and find ....
AND:
ii) the second given LINK:
https://www.researchgate.net/post/Why_is_Sucrose_added_to_lysis_buffer_in_some_dna_extraction_protocols
I just pointed to a possible source of explanations (haven't read much of the links given there, which at least confirms the reference [4] in WIKI ! ), but that Thread or Question is also contained in the search results (on RG) when searching for | lysis buffer sucrose | on RG
https://www.researchgate.net/search.Search.html?type=question&query=Lysis+buffer++sucrose
OK. Now the problem/ your request is still not finished or solved because there is no indication (neither in my explanations nor in literature I searched)
"why sucrose (and NOT glycerol or Glucose....) is added to lysis buffers for isolation of intact nuclei." (would be interesting to study the original recipe / technique / method and what hass been written there and if authors have an opinion on that)
As you say: "Also, in the context of nucleus, osmotic pressure is not really relevant, as the nuclear pores are huge." it might be that we ought not take into consideration tonicity issues... but 0.3 M Sucrose will increase osmolarity of the solution at least with + 300 mosmol. this will add considerably to hyperosmolarity of the whole lysis buffer solution (not to forget: The common isotonic buffer used in DNA isolation is buffered sucrose).
Last but not least I have to admit that I haven’t done isolation of intact nuclei in my former profession but sometimes had issues with density centrifugation of blood.
Other possibilities to elucidate the matter perhaps might be density and / or viscosity (and stickiness) issues.
NB: Have you found something in Sven's attachment "class_01_13_04.pdf"
(originally found @: https://www.uab.edu/proteomics/pdf_files/class_01_13_04.pdf ) ?
Best wishes too, Wolfgang
The other function is retention of isotonic osmolarity. The link (that somehow will not work, so I have added the file of Liz Brandon - UAB) has some info on the osmolarity of sucrose in comparison to other density gradient building options.
https://en.wikipedia.org/wiki/Lysis_buffer
https://www.researchgate.net/post/Why_is_Sucrose_added_to_lysis_buffer_in_some_dna_extraction_protocols
And now the downvoter can act again!
PS: @Sven: it might be possible that you forgot to insert the mentioned LINK. Perhaps you can do this : EDIT (click unto the "down-pointing arrow" next TOP right to your Name and affiliation line, find "EDIT", insert or correct Text AND DON'T forget to SAVE again (:-)
Thanks for pointing out the missing link (not evolutionary!). I have added the .pdf as the link will not copy.
Dear Sven,
But why sucrose, and not glycerol or any other substance. Also, in the context of nucleus, osmotic pressure is not really relevant, as the nuclear pores are huge.
Dear Wolfgang,
Considering that the wiki link does not contain any reference to sucrose and the other link is only very remotely related to my question, the downvoter action would seem legitimate, right?
Best wishes,
Lech
-Apologize for lengthiness-
Ok, dear Lech, Good morning.
Would you mind to provide the protocol (and/or source or Reference of the protocol) or at least a more detailed description of the lysis buffer you are using ??
Concerning the other matter:
i) the criticized WIKI-link:
Read in the WIKI-compilation:
ADDITIVES/OTHER:
Other additives include metal ions, sugar like glucose *) , glycerol, reducing agents like dithiothreitol (DTT).
*) and "sugar" might be also Saccharose=Sucrose: cf: reference [4]:
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/
find ADDITIVES (CTRL-F, insert | additives | Return ) and find ....
AND:
ii) the second given LINK:
https://www.researchgate.net/post/Why_is_Sucrose_added_to_lysis_buffer_in_some_dna_extraction_protocols
I just pointed to a possible source of explanations (haven't read much of the links given there, which at least confirms the reference [4] in WIKI ! ), but that Thread or Question is also contained in the search results (on RG) when searching for | lysis buffer sucrose | on RG
https://www.researchgate.net/search.Search.html?type=question&query=Lysis+buffer++sucrose
OK. Now the problem/ your request is still not finished or solved because there is no indication (neither in my explanations nor in literature I searched)
"why sucrose (and NOT glycerol or Glucose....) is added to lysis buffers for isolation of intact nuclei." (would be interesting to study the original recipe / technique / method and what hass been written there and if authors have an opinion on that)
As you say: "Also, in the context of nucleus, osmotic pressure is not really relevant, as the nuclear pores are huge." it might be that we ought not take into consideration tonicity issues... but 0.3 M Sucrose will increase osmolarity of the solution at least with + 300 mosmol. this will add considerably to hyperosmolarity of the whole lysis buffer solution (not to forget: The common isotonic buffer used in DNA isolation is buffered sucrose).
Last but not least I have to admit that I haven’t done isolation of intact nuclei in my former profession but sometimes had issues with density centrifugation of blood.
Other possibilities to elucidate the matter perhaps might be density and / or viscosity (and stickiness) issues.
NB: Have you found something in Sven's attachment "class_01_13_04.pdf"
(originally found @: https://www.uab.edu/proteomics/pdf_files/class_01_13_04.pdf ) ?
Best wishes too, Wolfgang
Hi, Lech,
a) of course you can use substitutes for sucrose with similar properties, but you must consider that b) many of these protocols were developed to allow isolation of various sub-cellular components after cell lysis in several consecutive steps, e.g. lysosomes, mitochondira etc., not only nuclei. So, many choices are historically rooted and as you know how conservative researchers can be: if a protocol works nicely, it will not be changed. As far as I know most of these protocols are from the 70s.
Dear Wolfgang,
Thanks for an elaborate reply. An example of a protocol involving a seemingly hypertonic buffer can be found in this paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653293/
Let me rephrase my question: what would be a buffer of choice for obtaining intact nuclei (nuclear fraction), preserving the content of nuclei (minimizing leakage of the soluble proteins), and in the same time efficient in lysing (breaking) cell membranes to break the cells open?
Best wishes,
Lech
@Lech Kaczmarczyk
Hi! Have you find any suitable protocol yet?
Best,
Sakib
DZNE Göttingen
Yes. I was using 0.25-0.32M sucrose. Seems to preserve nuclei better.
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