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difference between washing and resuspending a pellet?


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#1 kwong067

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Posted 26 January 2011 - 09:42 AM

what the difference between washing a pellet and resuspending a pellet? I'm doing protein purification.
Thanks!

#2 pito

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Posted 26 January 2011 - 10:11 AM

Washing is washing.. its cleaning the pellet by adding a product (dont know what you use?). You will not "move" or break the pellet , you just add a substance to clean the pellet (remove contamintions that are in your tube with the pellet).

Resuspending the pellet means that you resuspend the pellet , thus you make it go back in suspension: you "remove" the pellet (by removing I mean: you destroy the pellet, but its does stay in your tube? I hope this makes sense?)

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Radish

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Posted 26 January 2011 - 11:54 AM

what kind of protocol are you using?

Best regards

Radish

#4 leelee

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Posted 26 January 2011 - 09:02 PM

I've always taken washing to mean: resuspend in wash solution and then repellet.

#5 Radish

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Posted 26 January 2011 - 09:51 PM

usually I read washing as:

splash your pellet clean without breaking or ressuspending the pellet (that is also what I mean when writing a protocol).

Best regards
Radish

#6 leelee

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Posted 26 January 2011 - 11:35 PM

I'm actually certain that it is supposed to mean resuspend and then repellet.
I struggle to see how merely putting a solution over the top of a pellet and then aspirating it will sufficiently wash the cells/DNA/whatever. Surely you want to get in and around the cells and within the pellet to get rid of anything that could be trapped?

Eg. when washing a DNA pellet- kit instructions usually say something like this (taken from Machery Nagel booklet):

"Wash and dry DNA pellet
Add room-temperature 70 % ethanol to the pellet. Vortex briefly and centrifuge
at ≥ 15,000 x g for 10 min at room temperature (18 25 C)"

And on this page it is the same when referring to making competent cells....http://userpages.umbc.edu/~jwolf/m7.htm

"4. Wash the cell pellet in 250 ml of ice-cold WB as follows. First, add a small amount of WB to cell pellet; pipet up and down or gently vortex until cells are resuspended. Then fill centrifuge bottle with ice cold WB and gently mix. NOTE- the absolute volume of WB added at this point is not important."

And here on the BD page for staining cells for flow....http://www.bdbiosciences.com/support/resources/protocols/human_cyclins.jsp

"Wash cells once by resuspending the pellet in 30-40 ml of Wash buffer. Centrifuge at 200 x g for 10 min and aspirate supernatant."

#7 pito

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Posted 27 January 2011 - 01:14 AM

Leelee you are correct that most of the time "washing" also means "resuspending", but this is not always the case. Sometimes you can not disturp the pellet too much and you need to wash it by adding a solution and not resuspending* it (sometimes, you do need to vortex a bit or move the washing solution a bit with a pipette, but you can not break the pellet).

check for example:
http://www.chemagen....lood250_Kit.pdf

I think the problem is that washing is not a very well defined step and most often they mean: washing and resuspending, but that the writers of the protocol dont even bother anymore to add resuspending because 99% of the times washing means resuspending it also.

PS. often when doing DNA extraction you will need to add ethanol (second time) to the pellet and most often you add this ethanol without! resuspending the pellet: you simply add the ethanol, make the pellet come loose by vortexing gently and then you centrifuge it. But again, this depends on the people teaching you this: some mention not to disturb the pellet, other say you need to "dissolve" the pellet...





(*remember that DNA doesnt dissolve in ethanol... so when doing an ethanol wash step you dont dissolve the dna, you simply wash it , but you could also say you resuspend it , depending on the definition of resuspending.... Resuspending means to put back in suspension, not necessary disolve it... Its getting difficult now lol. But this is getting more on words and definitions.. and I am not a native speaker so, I wonder what people think about this that are native, english, speakers..)

Edited by pito, 27 January 2011 - 10:42 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#8 Radish

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Posted 27 January 2011 - 08:32 AM

Leelee you are correct that most of the time "washing" also means "resuspending", but this is not always the case. Sometimes you can not disturp the pellet too much and you need to wash it by adding a solution and not resuspending* it (sometimes, you do need to vortex a bit or move the washing solution a bit with a pipette, but you can break the pellet).

check for example:
http://www.chemagen....lood250_Kit.pdf

I think the problem is that washing is not a very well defined step and most often they mean: washing and resuspending, but that the writers of the protocol dont even bother anymore to add resuspending because 99% of the times washing means resuspending it also.

PS. often when doing DNA extraction you will need to add ethanol (second time) to the pellet and most often you add this ethanol without! resuspending the pellet: you simply add the ethanol, make the pellet come loose by vortexing gently and then you centrifuge it. But again, this depends on the people teaching you this: some mention not to disturb the pellet, other say you need to "dissolve" the pellet...





(*remember that DNA doesnt dissolve in ethanol... so when doing an ethanol wash step you dont dissolve the dna, you simply wash it , but you could also say you resuspend it , depending on the definition of resuspending.... Resuspending means to put back in suspension, not necessary disolve it... Its getting difficult now lol. But this is getting more on words and definitions.. and I am not a native speaker so, I wonder what people think about this that are native, english, speakers..)

I was going to give the DNA extraction example pito! LOL Bravo.

I usually state wash when there is some reagent that you really need to get rid off without getting it to reincorporate the pellet.
It is usually followed by another round of centrifuge before the actual ressuspension :)

Best regards
Radish




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