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.
Note: If you are interested in subsequent protein purification of membrane proteins follow THIS LINK to step 5 of this protocol.
Newly made observations indicate that by slightly modifying the protocol compared to a normal affinity chromatography, the protein yield can increase substantially.

See this protocol in action!

 
Solubilization of membrane protein using SMALP
  1. 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
  2. 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).
  3. Incubate the mixture at cold temperatures overnight (16 h, 4°C) while gently stirring/shaking the solution.
  4. 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.
  5. Every step from here on is about the especialities of membrane protein purification that come with the use of SMALPS and DIBMALPS.
    As handy as SMA and DIBMA are for membrane protein stabilization and solubilization, when it comes to the subsequent protein purification their overwhelming presence causes some problems. As figure 1 indicates that with increasing polymer concentration the protein yield of the purification assay decreases by a lot.


    Polymers influence on protein yield with concentration on protein purification Figure 1 Protein yield after batch purification with Nickel INDIGO affinity resin, in relation to polymer concentration after dilution. Increase presented as relative yield to purification with undiluted polymer (2.5 %).
    To avoid this decrease in protein yield, dilute your protein solution after solubilization by 1:10 or 1:20. Use your matching protein buffer for this solubilization. A standard buffer for this purpose would consist of 100 mM NaCl, 20 mM HEPES and with the pH adjusted based on the pI value of the protein.
  6. Perform the binding step of your protein purification protocol to overnight, not matter what your original protocol says. This is to give the beads time to bind all tagged protein in the increased volume. It is NOT necessary to increase the amount of beads used. Use the amount of beads that was meant for the volume prior to the 1:10 - 1:20 dilution.
  7. For the washing steps you can decrease the volume again after removal of the binding buffer. Through the buffer exchange the majority of the hindering unbound polymer should be removed. The following washing steps should be done with the normal protein buffer without the addition of polymer.
  8. Elution should be done following also with your your established protocols.
  9. Note: Protein quantification using UV absorbance spectrophotometry is hindered when using SMA. Take that into consideration.
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