SOP for High-Yield protein extraction and Enrichment

This SOP is based on the standard protocol from ProteoExtract Native Membrane Protein Extraction Kit Callbiochem I tailored it to enrich the membrane protein yield to make biomimetic nanoparticles.

Utility: The ProteoExtract™ Native Membrane Protein Extraction Kit has been developed for high-yield membrane protein extraction, providing 3-5 fold enrichment of integral membrane and membrane-associated proteins using non-denaturing conditions. It is designed for the isolation of native membrane proteins from a broad range of mammalian samples, including:

• Adherent tissue culture cells

• Suspension-grown tissue culture cells

• Frozen cell pellets

• Tissues

You can follow the detailed protocol for each condition here

The protocol in my case has been tailored to enrich membrane protein yield from murine and human monocytes/macrophages established cell lines, starting with at least 100 million cells, tweaking buffer concentration for the number of cells:

Buffer 1: ~ 1.2 mL for every 10 million cells

Buffer 2: 250ul for every 10 million cells

Range: subdivide your starting number of cells into 25–35 million cells per 50 ml falcon tube

Make sure the centrifuge is turned on and adjusted to 4◦C before beginning the experiment
Keep Wash and Extraction Buffers 1 and 2 on ice. While keeping the protease Inhibitor cocktail at room temperature.
Wash with 2mL of wash buffer. Mix the solution using a pipette, making sure that the solution is homogenous.
Transfer into an appropriate centrifuge tubes and sediment cells at 200g at 4◦C for 5 minutes. Remove and discard supernatant.
If 10 million cells no need to wash 2 times. One time is enough
If 20 million or more wash twice.
Pellets cells at 200g at 4◦C for 5 minutes. Discard the supernatant.
Add 10 ul of Protease Inhibitor to the wall of the tube and let it drop down to the pellet. Immediately add 1~1.2mL of buffer 1 to the tube. Mix carefully using a pipette. Incubate on a shaker on ice for 10 minutes.
Centrifuge at 16000g at 4◦C for 15 minutes.
Discard supernatant.
Add 5 ul of Protease inhibitor to the wall of the tube. Immediately add 200~250 ul of buffer 2 to the cell pellet. Mix and resuspend cells carefully using a pipette.
Incubate on a shaker on ice for 30 minutes. ( At this time start preparing standard curve dilution for quantification).
Centrifuge at 16000g at 4◦C for 15 minutes. (At this time finish preparing standard curve dilutions, have all wells filled, prepare dilution for sample, and make sure plate reader is turned on and ready).
KEEP the supernatant Transfer to a new tube and store on ice. Start the Quantification process.

Buffer 1: ~ 1 mL for every 10 million cells

Buffer 2: 250ul for every 10 million cells

Range: 25-35 million cells per tube

STARTING from 150 MILLION CELLS:

Make sure the centrifuge is turned on and adjusted to 4◦C before beginning the experiment
Keep Wash and Extraction buffers 1 and 2 on ice. While keeping the protease Inhibitor cocktail at room temp
Wash with 2mL of wash buffer. Mix the solution using a pipette making sure that the solution is homogenous
Transfer into an appropriate centrifuge tubes and sediment cells at 200g at 4◦C for 10 minutes. Remove and discard supernatant.
Wash with 2mL of wash buffer again.
Pellets cells at 200g at 4◦C for 10 minutes. Discard the supernatant.
Add 10 ul of Protease Inhibitor per tube (10ul x # of tubes = total amount of Protease Inhibitor to add) to the wall of the tube and let it drop down to the pellet.
Add about 2000uL of Buffer 1 into the original tube. Then divide 2000uL by how many tubes there are. Then add that amount of uL to each tube.
Subtract the amount added from 2000uL and fill each tube with buffer 1 to get total volume of 2000uL (2mL). Mix each tube carefully using a pipette. Incubate on shaker on ice for 10 minutes.
Centrifuge all tubes at 16000g at 4◦C for 15 minutes.
Discard supernatant.
Add 5 ul of Protease inhibitor (5ul x # of tubes = total amount of Protease Inhibitor to add) to the wall of the tube. Immediately add ~600ul-650 ul of buffer 2 to the cell pellet. Mix and resuspend cells carefully using a pipette. Then start adding all of tube 1 to tube 2, then add all of tube 2 to tube 3, and so on. Until all tubes are combined.
Incubate on a shaker on ice for 30 minutes. (At this time start preparing the standard curve dilution for quantification. Calculate how much Reagent is needed but DO NOT prepare at this point. Turn on the plate reader if you have time).
Centrifuge at 16000g at 4◦C for 15 minutes. (At this time finish preparing standard curve dilutions, have all wells filled, prepare dilution for sample, and make sure plate reader is turned on and ready)
KEEP the supernatant and store it in the new tube. Start Quantification process.

Brief protein quantification process:

Quantification of the extracted protein concentration was determined using a Pierce Rapid Gold BCA Protein Assay Kit according to the manufacturer’s protocol. Absorbance was measured at 480 nm on an FLUO star Omega microplate reader, and protein concentration was determined using a calibration curve with albumin diluted in 1× PBS at the following concentrations: 0, 25, 125, 250, 500, 750, 1000, and 1500 µg/ml.

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SOP for High-Yield protein extraction and Enrichment