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Large-Scale Purification of Non-Secreted Protein in E.Coli or CHO Cells

Large-Scale Purification

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#1 Pangea

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Posted 05 January 2013 - 03:38 PM

How can you lyse the cells if you have 1000 l or more? And purify from a Bioreactor?

#2 Curtis

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Posted 08 January 2013 - 09:38 PM

1000 is not much. A T75 flask of CHO cells would give you around 10^7 cells. I am sure a bioreactor gives much more than that. there are methods for lysing eukaryotic and prokaryotic cells. detergents play key role in that. look up for RIPA buffer. That's the most popular lysis buffer for eukarotic cells. For bacteria there are also many methods. depends on what you want to extract from them. Do you know Sambrook?

#3 Pangea

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Posted 09 January 2013 - 05:50 AM

Yes i know Sambrook and Maniatis. But you missed the sign behind 1000 . "Liters". But RIPA is quite a strong Lysis Buffer. However you say that there is no other technique to break the cells in high amount conc. than Lysis Buffer?

#4 Curtis

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Posted 09 January 2013 - 08:24 PM

We write liter with capital L. Like mL, uL.

Of course there are other methods, some people prefer to not use detergent, and instead break cells physically. there are machines for that. But in the lab most people use lysis buffers, because they are cheap. RIPA is really strong, and honestly it is not my favorite either. I used CHAPS buffer for some time, and then for my fractionation studies I used digitonin buffer. You can study different types of detergents at Wikipedia. I'd recommend you to study them carefully, it's important to know their differences.

1000 L is very large volume though, it's like 1 ton. it's a pilot scale. Even if you use lysis buffer you need to centrifuge that 1000 L to get a clear supernatant. that would need a huge centrifuge. I would also think for an alternative if I were you. I will check this and let you know because I have never lysed this many cells.

#5 Curtis

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Posted 09 January 2013 - 09:05 PM

here, I found these:

Posted Image

http://www.desmech.com/?p=28

This one claims they can even scale this up to industrial scale:
Posted Image
http://www.niro-soav...disruption.html

But I found this one more interesting, it works by nitorgen decompression:

http://www.zimmerman...ds/Parrcell.pdf

chemical and enzymatic methods would be costly for large-scale cell disruption, therefore companies use alternative methods. However they also have disadvantages, one of which is temperature rise.

#6 leelee

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Posted 09 January 2013 - 09:36 PM

Alert: Thread hijack ahead Posted Image

We write liter with capital L. Like mL, uL.


Actually, both are acceptable. Usually one would use lower case, but litre is the exception, where upper case can be used when concerned about mixing up a 1 and an l. And also, most countries spell it "litre". I'm pretty sure it's is just the Americans who use "liter".

(source: Internationional bureau for weights and measurements http://www.bipm.org/...apter5/5-1.html)

Edited by leelee, 09 January 2013 - 09:36 PM.


#7 Curtis

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Posted 09 January 2013 - 10:13 PM

:D @ leelee

#8 Pangea

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Posted 10 January 2013 - 11:17 AM

Thanks guy that gives me a better understanding how things are handled. Keep in touch.




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