I have read that I can use RIPA buffer for EV membrane disruption. Does this have to be followed by ultra centrifugation? Does anyone have a protocol for this?
Thanks.
Any strong surfactant will do. Sonication is also an option. You can also test your Bradford/BCA kit on your exosomes to see if the treatments make a large difference.
You can use physical methods (such as nitrogen cavitation, extrusion via porous membrane, or sonication) or chemical methods (detergents etc.). EV membranes are similar to cell membranes, so some methods to disrupt cell membranes can potentially apply
https://lab.plygenind.com/how-to-disrupt-extracellular-vesicle-membrane-in-order-to-study-their-content
Hi Maral Tabrizi
Yes, you can use RIPA (Radioimmunoprecipitation assay) buffer to disrupt extracellular vesicle (EV) membranes. It's a strong lysis buffer and it helps to solubilize proteins from EVs. However, keep in mind that RIPA buffer is not selective for EVs, so it will also lyse any cells in your sample.
Here's a general protocol you can follow:
Collection of EVs: Collect EVs from your desired source (cell culture supernatant, biological fluids, etc.) by differential ultracentrifugation or other EV isolation methods (like size exclusion chromatography, density gradient centrifugation, or commercial kits based on polymer precipitation).
RIPA Lysis: Resuspend your isolated EV pellet in RIPA buffer containing protease and phosphatase inhibitors. Typically, 100 µL of RIPA buffer is used for approximately 1x10^9 EVs but the exact volume can be adjusted based on your requirements. Incubate on ice for 30 minutes to 1 hour with intermittent vortexing.
Sonicate (Optional): Some researchers recommend a brief sonication step to ensure complete lysis.
Centrifuge: To remove insoluble material, centrifuge the lysate at ~14,000 x g for 10-20 minutes at 4°C. The supernatant contains your lysed EV proteins.
Protein Quantification: Use a protein assay (like BCA or Bradford) to determine the protein concentration in your sample.
Dear Dr. Maral Tabrizi
RIPA lysis buffer which contains a combination of both ionic and non-ionic detergents results in the highest number of identified peptides and proteins.
Yes, you may lyse EVs in an appropriate volume of RIPA lysis buffer. You may add protease and phosphatase inhibitors at the time of lysis.
You may use the following protocol.
You may use the following inhibitors in the RIPA lysis buffer at the time of lysis.
1 mM Sodium orthovanadate,
50mM NaF,
5 µg/ml Pepstatin,
5 µg/ml Aprotinin,
5 µg/ml Leupeptin.
Lysis may be performed on ice for 30min and placed in an ice-cold sonication bath for 30 s. This step may be followed by a gentle mix on ice for 15 min followed by centrifugation at 15000 g for 10min. Discard the pellet.
You may add 1 volume of 100% (w/v) TCA to 4 volumes of the sample, vortex and incubate for 5 min at 4°C. After centrifugation at 15000 g for 5min at 4 °C, the supernatant may be discarded, and the pellet may be washed with cold acetone. Centrifuge at 15000g for 2 mins at 4°C. Discard the supernatant. Repeat the washing step but this time using 80% cold acetone. Discard the supernatant. Air-dry the pellet (proteins) for 3 mins. You may dissolve the protein in the buffer of choice.
The protein content may be assessed using BCA assay.
If you wish to use the micro-BCA assay, you may add 5μL of sample to a 96-well microplate followed by 100μL of BCA reagent. Mix plate thoroughly on a plate shaker for 30 seconds. The plate may be incubated in the dark for 30min at 37 °C. The absorbance may be measured at 560nm, and protein concentration may be determined from a BSA standard curve.
Regards,
Malcolm Nobre
Hi Saman Behboodi Tanourlouee and Malcolm Nobre I appreciate both of your explanations in the use of EV lysis prior to BCA, I notice that you both recommend centrifugation after lysis, why is this required, I have read other papers that don't mention this, thank you for your input!!Article Comparison of methods to isolate proteins from extracellular...
andArticle Exosome isolation from distinct biofluids using precipitatio...
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