Hi there,

Urea is a very efficient solubilzing agent allowing the prevention of protein agregation. It is usually used at 6M-8M in extraction and loading buffers. The main drawback is that urea has to be present in every lane of the gel to avoid lateral diffusion of urea containing samples towards the lanes devoided of urea.

Hi,

If I use SDS-PAGE gels containing urea, will it help?

Hi again,

No need to include urea within the gel as long as the samples you load contains high concentration of it.

Hi: Here some useful papers:

http://tools.thermofisher.com/content/sfs/manuals/MAN0011863_MemPER_Plus_Memb_Protein_Extract_UG.pdf

https://link.springer.com/protocol/10.1385%2F1-59259-655-X%3A283#page-1

http://www.embl-hamburg.de/workshops/2006/PEPC5/talks/Andy.pdf

http://onlinelibrary.wiley.com/doi/10.1002/elps.200305387/pdf

http://onlinelibrary.wiley.com/doi/10.1002/0471143030.cb0101s05/pdf

90

Hi Yen

See the reference in attachment 1

https://onlinelibrary.wiley.com/doi/10.1002/elps.200305387/pdf

2

https://www.ncbi.nlm.nih.gov/pubmed/20978985

3

https://grad.md.chula.ac.th/english%20old/data/PEP_2011_8_membrane_protein_secure.pdf

4

https://it.science.cmu.ac.th/ejournal/dl.php?journal_id=274

5

http://www.unr.edu/proteomics/sample-preparation/2d-protocols/protocol-protein-fractions

81

6

https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/solubilisation_membrane_proteins/

92

7

http://www.abcam.com/plasma-membrane-protein-extraction-kit-ab65400.html

Hi, I think I will try with urea first. Will update the results later.

105

Good Luck

I have described how to develop solubilisation protocols for membrane proteins in chapter 15 of Biophysical Chemistry of Proteins, Springer 2011.

I have no access to the book. do you mind to share that particular chapters here?

Ok, here is the LaTeX-Source for this part of the book:

\subsection{Developing a solubilisation protocol}\index{solubilisation|(}

If you need to develop a procedure for membrane protein isolation, you can estimate the amount of detergent required for solubilisation by the following considerations:

There should be at most one protein molecule per detergent micelle\index{micelle!to protein ratio}. Since the number of protein molecules per micelle will be (in first approximation) \Name{Poisson}-distributed

\index{Poisson@\textsc{Poisson} distribution}, you need about \num{10} detergent micelles per protein molecule. For the average molecular weight of membrane proteins you can assume \SI{100}{kDa}, thus a \SI{1}{mg/ml} protein preparation is about \SI{10}{\micro M}, and you need a micelle concentration of \SI{100}{\micro M} to solubilise them.

One detergent

\index{detergent!concentration required for solubilisation} micelle can solubilise about \num{10} lipid molecules. In first approximation you can assume that a preparation containing \SI{1}{mg/ml} membrane proteins will be also about \SI{1}{mg/ml} in lipid (actually somewhat more), with an average molecular mass of \SI{750}{Da}. Thus the \SI{1}{mg/ml} protein preparation will be about \SI{1.3}{mM} in lipid and you need a micelle concentration of \SI{130}{\micro M} to solubilise them.

To solubilise both lipid and protein from a \SI{1}{mg/ml} membrane protein preparation, you need a micelle concentration of \SI{230}{\micro M}. The next thing you need is the number of detergent molecules per micelle (aggregation number\index{detergent!aggregation number}, \skalar{\bar m}), the molecular mass of the detergent and its critical micellar concentration\index{cmc}. These data you can find in the literature of chemical supply companies. For example, \acs{CTAB}\index{CTAB!in solubilisation} has a molecular mass of \SI{364.5}{Da}, a \acs{cmc}\index{cmc!and solubilisation} of \SI{1}{mM} and \skalar{\bar m} $\approx$ \num{170} detergent molecules per micelle.

To get a micelle concentration of \SI{230}{\micro M}, you need \skalar{\bar m} * \SI{230}{\micro M} + \acs{cmc}, that is \SI{40.1}{mM} in this case, or \SI{14.9}{mg/ml}.

This calculation uses a lot of handwaving, but gives quite reasonable results (see fig. \ref{fig:solub}). The next step is to test things in the lab. For this purpose, do small scale solubilisation experiments (about 150--\SI{200}{\micro l}) with \num{0.25}, \num{0.50}, \num{1.0}, \num{2.0} and \num{4.0} times the calculated detergent concentration. Stir the samples on ice for \SI{60}{min}, take an aliquot for enzyme\index{enzyme!membrane} activity determination and spin the reminder for \SI{100000}{\AbsVec{g}} for \SI{30}{min} (desktop-ultracentrifuge\index{ultracentrifugation} with rotor for \SI{0.2}{ml} tubes). Determine the protein concentration and the enzymatic activity in both the supernatant and the pellet. It can be useful to repeat the activity determinations after a weekend, to account for the time required later for purification. Repeat this experiment for a number of different detergents.

What you would like to find is a detergent that is able to almost completely solubilise your protein (ratio of protein amount in solution/pellet) without inactivation (enzymatic activity in the aliquot taken before centrifugation). Osmolytes like sucrose\index{sucrose!as osmolyte} or glycerol

\index{glycerol!as osmolyte} can protect proteins during solubilisation. Proteins may be more sensitive to proteolytic attack in the solubilised state, appropriate protection is required (\Foreign{vide supra}). In addition, alkylamines

\index{alkylamine!in solubilisation} and polyamines\index{polyamine!in solubilisation} can increase the solubilisation yield \citep{Yas-10} (about \SI{100}{mM}), probably by destabilising lipid membranes. They should be removed as soon as possible after solubilisation however, as they can denature proteins upon prolonged contact.

In practice you will find that small detergent\index{detergent!selection of} concentrations tend to activate membrane enzymes\index{enzyme!membrane}, this may be caused by increased membrane fluidity\index{membrane!fluidity}. As the detergent concentration increases, inactivation is often seen. So you need to find a detergent that can solubilise your protein at a concentration where it is not yet inhibitory.

\begin{figure}

\caption{Solubilisation of Mdr1\index{Mdr1 (ABCB1)} (P-glycoprotein, an ABC-type transport ATPase\index{ATPase!ABC-type}) with two different detergents, cetyltrimethylammonium bromide\index{CTAB!in solubilisation} (CTAB, left) and octyl glucoside\index{octyl glucoside} (OG, right). In both cases the addition of small concentrations of detergent initially increases enzyme\index{enzyme!membrane} activity (green dashed lines). However, increasing \acs{OG} concentration leads to a rapid inactivation of \acs{Mdr1}. \acs{CTAB} leads to a much slower decline in \acs{ATPase} activity, solubilisation (red solid lines) can be achieved at concentrations that do not cause substantial inactivation. The concentration calculated to achieve solubilisation are marked by arrows. }

\label{fig:solub}

\centering

\includegraphics[width=\textwidth]{pdf-files/solub}

\end{figure}

For example, the following results were obtained with different detergents for the solubilisation of \acs{Mdr1}\index{Mdr1 (ABCB1)} (for other proteins, results may be completely different):

\begin{description}

\item[total inactivation even at low concentration:]{\acs{SDS}\index{SDS!effect on Mdr1}}

\item[inactivation at solubilising concentration:]{\acs{CHAPS}\index{CHAPS}, Zwittergent\index{Zwittergent}, cholate\index{cholate}, desoxycholate\index{desoxycholate}, dodecylmaltoside\index{dodecyl!maltoside}, octyl glucoside\index{octyl glucoside}, Hecameg\index{Hecameg}, Lubrol WX\index{Lubrol WX} , Tween 20\index{Tween 20}, Brij 35\index{Brij}}

\item[at least \SI{100}{\%} activity at solubilising concentration:]{\acs{CTAB}\index{CTAB!in solubilisation}, Tri\-ton-X100\index{Triton-X100}, C$_{12}$E$_8$\index{C12E8@C$_{12}$E$_8$}, C$_8$E$_5$\index{C8E5@C$_8$E$_5$}}

\end{description}

These experiments have told you which detergents can be used at which concentration to solubilise your protein without (too much) damage. In the next step, try to add lipid\index{lipid} (and more detergent), until you can separate your protein from others (\Foreign{i.e.}: each protein is in a single micelle). Rate zonal centrifugation on glycerol \index{glycerol!in ultracentrifugation}- or sucrose gradients is a simple method to use, if you analyse your fractions by \acs{SDS}-\acs{PAGE}\index{SDS!PAGE}, your protein should be enriched from proteins of lower and higher molecular mass in some fractions. Alternatively, you can incubate the solubilised protein with glutardialdehyde\index{glutardialdehyde} and look for cross-linking by SDS-PAGE or SEC-HPLC\index{HPLC}. Cross-linking indicates that there is more than one protein per micelle, and that the detergent/protein-ratio needs to be increased \citep{Mai-07}. For \SI{1}{mg/ml} protein, the addition of \num{2}--\SI{3}{mg/ml} lipid should usually suffice.

Note that in all buffers and gradients you need to include your detergent at the \acs{cmc}\index{cmc}, to maintain the protein/detergent/lipid micelles. The \acs{cmc} however depends on the composition of the buffer and should be determined as described above. \index{detergent|)}

\index{detergent!in membrane solubilisation|)} \index{solubilisation|)}