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Large protein removal (fractionation)


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6 replies to this topic

#1 TurtleBob

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Posted 21 December 2014 - 08:20 PM

Hi All, 

 

I could really use some help here and would appreciate any direction!

 

I have been trying for a while to detect BDNF protein (mature form is 14kda as a monomer).

It is present in very low levels in skeletal muscle. Since muscle contains many other larger and more abundant proteins, I would like to fractionate the muscle homogenate to retain proteins below ~50kda. How can I do this?

 

Is there anything similar to the chemical method of acetonitrile for removing albumin from serum?

This report suggests that acetonitrile might be good for general removal of large proteins (

Any thoughts on this? 

 

Does anyone know of any other method, chemical or physical? As BDNF is susceptible to degradation, I would prefer to try something that more crudely removes large proteins in less steps (rather than more thoroughly in more steps). Just something to get that smaller protein fraction of muscle homogenate to enhance the proportion of BDNF...

 

Thank you, thank you for any suggestions!

 

TurtleBob



#2 TurtleBob

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Posted 21 December 2014 - 08:21 PM

Sorry - here is the link http://www.abrf.org/...2003/Alpert.pdf



#3 mdfenko

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Posted 22 December 2014 - 04:16 PM

how about gel filtration or membrane filtration?


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genius does what it must
i do what i get paid to do

#4 phage434

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Posted 22 December 2014 - 06:57 PM

I'm a fan of the Microcon or Centricon filters (Millipore). A 20 kD version would probably be a good choice to retain large proteins and to pass your smaller ones.



#5 mdfenko

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Posted 23 December 2014 - 02:26 PM

14kd may not make it through a 20kd membrane. a 30 or 50kd membrane may be better.


talent does what it can
genius does what it must
i do what i get paid to do

#6 TurtleBob

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Posted 24 December 2014 - 11:41 AM

Great! Thank you both so much. The Microcon filters look pretty straight forward to use. I might start with this. this isn't really my "strong" area.. so another reason simpler is better.

Thanks again, and happy holidays!



#7 labtastic

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Posted 29 December 2014 - 10:29 AM

I am not a fan of using microcon filters for separating proteins based on size. Depending on how your protein behaves (oligomers, non-specific binding to membrane, etc), there is a good chance it wouldn't flow thru a 100kDa filter. I've seen monomeric GFP at 28kDa not go thru a 100kDa filter. You will just have to try and see but don't be surprised if you don't see your protein in the flow thru.

 

Your best bet is gel-filtration.

 

There are other simple forms of fractionation as well that aren't based on size necessarily but could remove a large proportion of contaminating proteins. Stepwise ammonium sulfate precipitation could work well. Depending on the pI of your protein, you could send it through a cation or anion exchange column (i.e. if your protein is negatively charged, your protein should pass through a cation column while removing everything positively charged). 






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