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SDS removal through dyalisis

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chululay
Argentina

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Topic Started by chululay
on 11/6/2008 5:57 AM   
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Hi! I´m trying to remove SDS from my protein sample, since I use SDS to extract my proteins. Now I want to treat my cells with the protein extract, but it has SDS, so it lyses the cells.
I tried to remove SDS through dyalisis, but SDS is still in my sample.
Couls you help me, please?
Thanks in advance!


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R Bishop
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Posted By R Bishop
on 11/6/2008 6:18 AM   
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That question comes a bit. The classic way to remove SDS from protein samples is by acetone precipitation of the protein, which leaves the SDS in the soluble phase. That method is not perfect though since crashing the protein out of solution can casue problems with activity and aggregation. However there is an excellent alternative. Pierce (now ThermoFisher) makes a small volume SDS removal kit that is pretty cheap at $130 US.

Here's the link to the SDS-Out SDS Precipitation Reagent
pierce SDS-Out Precipitation kit


I've used it a couple of times prior to mass spectrometry with good success. Can you let us know if you have success with your sample?

Rusty Bishop

"Everything should be made as simple as possible, but no simpler."
-- Einstein


Last edited Nov 06, 2008, 8:19 AM by R Bishop

chululay
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Posted By chululay
on 11/6/2008 6:59 AM   
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Thank you very much!
Do you know if it is possible to remove SDS through dyalisis? May be my protocol was inneficient.
I dyalized against PBS with 2 changes, the last one over night.



R Bishop
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Posted By R Bishop
on 11/6/2008 9:08 AM   
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The answer is effectively no. It cant be. Here's a quick blurb on why.

Sodium dodecyl sulfate (SDS) and SDS-Lauryl have a polar anionic sulfate group at one end of their structures and a straight chain nonpolar region at the other end. The dual polarity of SDS allows it to solubilize proteins by imitating their structure. The CMC of SDS is dependent on salt concentration. The CMC for SDS is 8.0 mM in water, 3.5 mM for 10 mM NaCl, and 1.4 mM for 100 mM NaCl in water. Although SDS has a high CMC and a low CMC molecular weight, it tends to bind tightly to cationic molecules because of its anionic nature. SDS that is bound to molecules cannot be removed by dialysis.

taken from this

link

Rusty

"Everything should be made as simple as possible, but no simpler."
-- Einstein



chululay
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Posted By chululay
on 11/6/2008 9:30 AM   
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Thank you so much!
I will try acetone precipitation.
Best regards,
Lucía.



R Bishop
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Posted By R Bishop
on 11/6/2008 9:58 AM   
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Sounds good. If you find a good protocol can you post it here?

BTW - What does chululay mean?

Thanks

Rus

"Everything should be made as simple as possible, but no simpler."
-- Einstein



chululay
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Posted By chululay
on 11/6/2008 13:28 PM   
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Sure! I will post it when I find the best way to remove SDS from my sample.
Chululay is just the way my cousin called me when we were children, it means anything.
Thaks for your help.



chululay
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Posted By chululay
on 11/19/2008 6:30 AM   
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I`m trying to remove SDS from my protein sample through acetone precipitation. After precipitation is very difficult to dissolve protein pellet in PBS. Do you know a way to dissolve the proteins?
Thanks!



parvoman
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Posted By parvoman
on 11/26/2008 22:23 PM   
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I had this problem a long time ago and I remember that we changed to a different precipitation protocol. I'm not 100% sure but I believe it was a TCA precipitation followed by two washes of the pellet with Acetone containing HCl then two washes with just acetone. Briefly air dry until the last visible drops of acetone have evaporated. Resuspend in buffer.

If I were you though, I'd go for the kit that was recommended by R Bishop, especially if you need to retain protein activity.

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chululay
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Posted By chululay
on 11/28/2008 8:31 AM   
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I finally eliminated SDS from my protein sample by acetone precipitation. I disolved the pellet using a syringe and a needle 25G. My protein was intact as a could prove it by SDS PAGE.Thank you very much for your kind advice!



gashok1981
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Posted By gashok1981
on 1/9/2009 2:26 AM   
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Hi Chululay,
SDS can be removed from the sample good enough by dialysis, but you have to choose the dialyzing solvent in mild acidic condition (adjust the of Distilled water/saline to the pH 4.0-4.5). I had optimized this technique for my cell lysate. I never say that acetone precipitation will not be a suitable method it is very fastest one, but you may not know the lose molecule in recovery.



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