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<p class="MsoNormal"><span lang="EN-US">Dear Teige<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">we have quite intensely used SMAs for the extraction of a highly abundant protein from native membranes. Compared to extraction with detergents, SMA-extraction seems to be much milder and preserves the active state of
our target protein, possibly mediated by the preservation of the native environment (lipids, binding partners). As with nanodiscs, it is probably wise to try several protein/polymer combinations – unfortunately no ‘magic bullet’ here.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">We have tried XIRAN 30010 (that we got as a free sample from Polyscope ~100 mL) and Sokalan CP9 (also a free sample from BASF – smallest sample size is 1 liter!).
<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">We have dialyzed the solutions against the desired buffer, as the pH of the original solutions (ca. 30% v/v) is 10-13!.
<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">XIRANS are well behaved and after dialysis you reach the desired pH. In contrast to that, Sokalan never reaches the desired pH and stays one pH-unit above that (despite >24 h dialysis and several buffer changes) – we
are not sure, why this is the case. Possibly it has some buffering capacity by itself??<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">When using SMAs/DIBMAs be careful with higher concentrations of divalent cations (> 1 mM), as they might change the solubility of the polymers themselves and lead to precipitation. This is less an issue for Sokalan. Also
the pH-range is limited (we used pH 6.5 and 7.5), but you may have to try it out.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">Also binding of your protein to NiNTA-matrices can be an issue in some cases.
<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">We also always omitted glycerol in the extraction step, but this is no must (compare the recent AcrB structure). The extraction parameters have to be established for every protein/polymer combination. We used a 0.25-8%
(v/v) range for 30-90 min for initial small-scale tests. The efficiency was a bit lower than with detergents, but it is all about quality, right?<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">We are using ash-free filter paper for blotting/plunge freezing, as the residual divalent cations may lead to precipitation at the very last step – but I would consider this to be rather “voodoo magic”. We never saw any
“weird” behavior of SMALPs in plunge freezing using holeys (with Vitrobot and Leica blotters).
<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">In contrast to detergent-solubilized protein, boiling of SDS-samples with XIRAN-solubilized proteins is easily possible without inducing the “laddering effect” and thus artefacts. Sokalan-solubilized protein smears in
gels. We tried the CH3OH/CHCl3/H2O-extraction method (which is in principle a Wessel-Fluegge protein precipitation) as suggested by Sandro Keller, but in our hands it did not work so well. This might be a protein-dependent behavior.
<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">In the end we went for nanodiscs in our project, as the SMALPs appeared somewhat roundish/blobby in cryogenic conditions (our target protein is rather small with ~250 kDa and it is quite unclear how large the SMALP-disks
really are). For larger membrane proteins this might not be an issue.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">A huge knowledge base is found under: <a href="http://www.smalp.net/">
http://www.smalp.net/</a>. There you can find some of the tips I have mentioned above and citations of most SMALP-related publications.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">There was a SMALP community meeting recently with contributions from most of the experts in the field: http://www.smalp.net/SMALP-2018-agenda.pdf
<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"> </span></p>
<p class="MsoNormal"><span lang="EN-US">All the best,<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">Magdalena<o:p></o:p></span></p>
<!--EndFragment-->
<div><br>
<div style="font-family:Tahoma; font-size:13px"><font face="Arial" size="2"><br>
--------------------------------------------------------------<br>
Dr. Magdalena Schacherl<br>
Structural Dynamics of Proteins<br>
<br>
phone +(49)228-9656-278<br>
e-mail magdalena.schacherl@caesar.de<br>
<br>
research center caesar<br>
center of advanced european studies and research<br>
- an associate of the Max Planck Society<br>
<br>
Ludwig-Erhard-Allee 2<br>
53175 Bonn, Germany<br>
www.caesar.de</font></div>
</div>
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<hr tabindex="-1">
<div id="divRpF860911" style="direction: ltr;"><font face="Tahoma" size="2" color="#000000"><b>Von:</b> 3dem [3dem-bounces@ncmir.ucsd.edu]" im Auftrag von "Matthews-Palmer, Teige Rowan Seal [t.matthews-palmer14@imperial.ac.uk]<br>
<b>Gesendet:</b> Dienstag, 12. Juni 2018 18:04<br>
<b>An:</b> 3dem@ncmir.ucsd.edu<br>
<b>Betreff:</b> [3dem] Any experiences with SMA extracting proteins in native membrane?<br>
</font><br>
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<div></div>
<div>Dear 3DEMers,
<div class=""><br class="">
</div>
<div class="">I only recently became aware of SMA co-polymers for solubilising membrane proteins in a ‘native bilayer’ environment
<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593810/" class="" target="_blank" rel="noopener noreferrer">
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593810/</a> - and to be honest was a bit shocked I hadn’t heard of this option before. </div>
<div class="">Supposedly you can directly solubilise your target with some amount of native membrane, which seems an improvement over MSP nanodiscs and salipro which require you to transfer your detergent-solubilised protein in to the lipid construct.</div>
<div class=""><br class="">
</div>
<div class="">I wonder how many of you have experience with this. I’d love to hear anyone’s first hand experience and any caveats that may be hard to find in the literature - like solubilisation efficiency, dependence on your target’s density in your membranes,
any difficult behaviour with plunge freezing on holey support foils? Importance of testing different co-polymer average sizes to suit your target?</div>
<div class=""><br class="">
</div>
<div class="">Thanks & best wishes,</div>
<div class="">Teige</div>
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