Membrane proteins (MPs), part of biological membranes, play a crucial role in basic cellular structure and functions, including cell integrity, signal transduction, molecular recognition, material transport, and cell-to-cell communication. Additionally, MPs are the largest category of drug targets. More than 60% currently available therapeutic molecules target one or more MPs. However, there are relatively few MPs with known crystal structures due to the technical challenges associated with membrane protein extraction, solubilisation, and purification.
MPs are divided into two major classes: integral membrane proteins (IMPs) and peripheral membrane proteins (PMPs). IMPs are permanently attached to the membrane lipid bilayers. While, PMPs are temporarily associated with either the lipid bilayer or IMPs by means of non-covalent interactions. Generally speaking, IMPs can be purified by more stringent techniques than PMPs, whose extraction just needs a high PH buffer. Here we provide a protocol for IMPs extraction focusing on homogenate preparation, soluble proteins removal, membrane proteins extraction and detergent removal.
1. For cultured cells: collect cells (0.2-1 x 108) and wash with ice-cold PBS. Centrifuge at 500 x g for 5 min at 4°C and aspirate the PBS. Resuspend the pellet in 2 mL ice-cold homogenization buffer in a mechanical homogenizer. Homogenize cells on ice. Then skip to Step 3.
2. For tissues: weigh a certain amount of tissues and mince into pieces on ice. Wash with PBS, centrifuge at 500 x g for 5 min at 4°C and discard the wash buffer. Homogenize tissues on ice in 2 volumes of the homogenize buffer, until it is completely lysed.
3. (Optional) Sonicate sample using two 10 second pulses (30 seconds in between pulses) using a probe sonicator. Keep sample in an ice bath and keep probe away from the sample-air interface to minimize foaming.
Soluble proteins removal
4. Transfer the homogenate to a fresh 1.5 mL Ep tube and centrifuge at 700 x g for 10 min at 4°C to remove the intact cells, nuclei and cell debris.
5. Collect the supernatant and discard the pellet.
6. Centrifuge the supernatant at 100,000 x g for one hour at 4°C.
7. Carefully aspirate the supernatant (containing cytosol fraction) and collect the pellet.
8. Wash the pellet with homogenization buffer and re-centrifuge at 100,000 x g for one hour at 4°C. Collect the pellet.
Membrane proteins extraction by Triton X-100
9. Resuspend the pellet in 1 mL homogenization buffer.
10. Add cells drop-wise to the 2% Triton X-100 while stirring
11. Incubate for 30 min at 4°C with occasional vortexing.
12. Centrifuge at 100,000×g for 30 min at 4°C.
13. Transfer the supernatant to a fresh tube.
Note: For peripheral membrane proteins fractionation, you just need resuspend the pellet (obtained in Step 8) in high pH buffer (100 mM Na2CO3, pH 11.3). Incubate for 30 min at 4°C with occasional vortexing and centrifuge at 100,000 x g for one hour at 4°C. Collect the supernatant.
Detergent removal by adsorption chromatography
Note: Before starting, ensure that your detergent is non-ionic detergent (e.g. Triton X-100) and the molecular weight of protein is large enough to avoid entrapment in the pores of the absorption matrix.
14. Apply distilled water through the column matrix, followed by blocking buffer.
15. Wash the column with wash buffer.
16. Repeat Step 15.
17. Transfer the supernatant (obtained in Step 13) to the column matrix.
18. Collect the protein fractions for analysis.
|1.||Smith SM. Strategies for the purification of membrane proteins[J]. Protein chromatography: methods and protocols, 2011: 485-496.|
|2.||Wiśniewski J R. Protocol to enrich and analyze plasma membrane proteins from frozen tissues[J]. Organelle Proteomics, 2008: 175-183.|