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adding Tween80 to bacterial culture for protein purification ?!


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#1 M_shabani_medbiotech

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Posted 10 June 2013 - 05:43 AM

hi ,

my supervisor just asked me to add 0.03 % tween80 to my bacterial culture which i induced by IPTG , he believes that it helps the proteins trapped in membrane be secreted easily so i will be able to extract them from the supernatant after centrifuging bacterial cells .

has any body heard any thing about this ? what is tween for ? doesn't it have negative effects on my cells and my recombinant protein ?!

thanks in advance ...
Mina

#2 labtastic

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Posted 10 June 2013 - 06:07 AM

I'm a little confused as to at what point in time you are adding the Tween. Are you adding it to the culture itself while inducing with IPTG? If this is the case, I would not recommend. it.

I suspect though you mean add it to the buffer that you resuspend your cell pellet and the lyse the cells in? If that is true, then yes your advisor's idea is reasonable. "Sticky" (i.e. hydrophobic) proteins can often associate with membranes and themselves when over expressed to form less soluble aggregates, which can be pelleted when you centrifuge out the membranes. Adding a little detergent to the extraction buffer can help minimize this by disrupting membrane-protein interactions, thereby making your protein more soluble.

If your protein has an affinity tag for purification, the tween can easily be removed by thoroughly washing the resin with detergent-free buffer.

#3 M_shabani_medbiotech

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Posted 10 June 2013 - 08:09 AM

actually i added it to culture media Posted Image 2 hours after i had added IPTG . tomorrow i'm going to perform ELIZA to detect my recombinant protein ! OH GOD , have i screwed up ?!!Posted Image Posted Image

#4 labtastic

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Posted 19 June 2013 - 08:44 AM

I haven't heard of anyone adding detergents to a culture during expression, but that doesn't mean it can't be done. One thing I've learned working with proteins is there are no rules...only suggestions and trends. There is only one way to find out if the tween does anything. Try it first small scale. No sense in growing buckets of culture with tween if you're not sure of the effect.

Having said that though, I would also recommend trying other things like changing the induction temperature, media, cell line, expression construct, vector, etc. These things can make dramatic differences in expression levels.




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