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Protein purifcation without chromatography


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6 replies to this topic

#1 2xzwei

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Posted 23 April 2013 - 12:10 AM

Hi!
I've managed to design bacteria that are able to secrete a protein out of the interleukin family. Unfortunately this protein has no HIS-tag etc. for positive selection.
Because the secreted amout of protein is very low (12ng/mL) I have to do a purification/concentration step which I have never done bevor. I did some reading and for the most part people use chromatography to get their protein. Are there any other (simpler and low-price) methods that one can do in the lab?
Thanks in advance

#2 bigudukaz

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Posted 23 April 2013 - 12:52 AM

I think the best choice would be using the spin columns to get proteins and maybe chromatography after to get as less of extra protein as possible. as the concentration is very low - spin columns, at least i think so, would be your best choice as you can get fairly big ammount of medium threw it and upconcentrrated proteins to fair ammount. It should work as your bacteria is secreting it and you dont need to digest the it :)

#3 2xzwei

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Posted 23 April 2013 - 02:06 AM

Thanks for this suggestion! So how does this work - just put the sample on the column where all protein will be retained and then I can elute it? I've never done like this before.

Would it be reasonable to prior concentrate the proteins by ammonium sulphage / TCA precipitation?

#4 mdfenko

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Posted 23 April 2013 - 04:23 AM

you can partially purify by differential solubility with various precipitants (eg ammonium sulfate, acetone, ethanol).

tca precipitation will denature the protein (sometimes somewhat reversible). this may be okay if you are not concerned with the functionality of the protein. tca will precipitate (nearly) all proteins at a relatively low concentration, so it won't be useful as a purification step (only for concentrating).
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#5 HOYAJM

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Posted 23 April 2013 - 07:08 AM

I'm not sure spin columns will be the best choice for this. You have a very small yield. I believe most spin columns claim they can recover ~90% of your proteins. But we all know the %recovery values can not always be matched. I would just worry that the little protein you have left would go through the column and you may be left with 2ng/mL. And at that point you have essentially zero protein of interest. Maybe dialysis could work? It depends on the size of the contaminant proteins in relatiion to your protein of interest.

#6 2xzwei

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Posted 23 April 2013 - 07:29 AM

So I just did this preliminary test in 12-well plates with few bacteria (3mL total volume which gave about 12ng/mL desired protein). I'm going to repeat this in larger scale (200mL culture) and hopefully the protein yield will be much higher...

The spin columns I found had only very low sample volumes (~100µL). Would it work to 1st precipitate the proteins with ammonium sulphate, dissolve the pellet in buffer and then go for spin columns?

Edited by 2xzwei, 23 April 2013 - 07:29 AM.


#7 mdfenko

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Posted 23 April 2013 - 10:46 AM

after dissolving the pellet from ammonium sulfate precipitation you will have to dialyze away residual as.

avoid the spin filter by suspending the pellet in a low volume.
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