Solubilization of membrane proteins using the synthetic polymer styrene-maleic acid or SMA
This protocol discribes how the solubilization of membrane proteins with SMA works. It applies to all of our SMA products and includes small suggestions for modifications for several parts of this protocol if the standard variant does not work.
|Solubilization of membrane protein using SMALP|
SMA allows for reconstitution of membrane proteins during various steps of purification. It is used with:
- whole cell suspensions
- supernatant of centrifuged cell lysate (9,000 x g)
- pellets of centrifuged supernatant (100,000 x g)
Note: Besides 50 mg SMA the lyophilisate contains Buffer adjusted to pH 7.5 for stabilization
Table 1: Volume of the protein sample to adjust a 50 mg SMA sample to final concentrations recommended for screening purposes.
|Volume [mL]||SMA (w/v)|
|12,50 ||0,4 % |
|7,14 ||0,7 % |
|5,00 ||1,0 % |
|3,33 ||1,5 % |
|2,50 ||2,0 % |
|2,00 ||2,5 % |
|1,67 ||3,0 % |
|1,43 ||3,5 % |
|1,25 ||4,0 % |
Add the protein solution directly to the ready-to-use lyophilized SMA powder and invert/vortex the tube until the SMA is completely dissolved. It is advised to use 2 mL of your protein solution with 50 mg lyophilized SMA to get a final concentration of 2,5 %. To further optimize your solubilization different SMA concentrations within the range of 0,4 % - 4 % are recommended (Tab. 1).
Incubate the mixture at cold temperatures overnight (16 h, 4°C) while gently stirring/shaking the solution.
Collect a sample for Western Blot analysis. Separate solubilized protein from insolubilized protein and debris with a centrifugation step (100 000 x g, 1 h, 4°C). Draw a sample of the supernatant to validate the solubilisation through Western Blot analysis and save the rest for further use.
Note: Protein quantification using UV absorbance spectrophotometry is hindered when using SMA. Take that into consideration.