Hallo, please can i ask you... is it possible to use only sonication instead of RIPA buffer to prepare the sample (isolation of heart mitochondria) for LC/MS analysis? When using RIPA is it necessary to purify the sample through the SPE columns C-18 in the finalization, isn`t it? Would we like to avoid this, is it possible to use only sonication instead? Thank you for your response!
That depends on the composition of your RIPA (radio immuno precipitation assay) buffer. The "standard" components are 20 mM Tris-HCl, pH 7.5, 150 mM NaCl. In addition, EDTA (metallo-protease inhibitor), other protease inhibitors, NaF (enolase inhibitor, bacteriostatic), and detergents (NP40, DOC, SDS) may be added.
So you can simply leave out anything that interferes with your assay, if the function of that substance is not required.
I’d be asking why you are using RIPA buffer at all? If you are ultimately analysing your samples by LC-MS, you need to start with a purification protocol that is MS compatible. There are numerous protocols available, all that will happily lyse and solubilise your samples and are compatible with sonication and ultimately LC-MS analysis. Search the literature for more information specific to your requirements.
Sonication will assist in membrane disruption with your mitochondrial sample, but it alone would be less effective in extracting membrane-associated protein. The detergents within RIPA (or similar lysis buffers) are there to break down the membranes and solubilize membranous protein content. If your LC/MS goal is to include membranous protein, you will need something in your buffer to help solubilize them. Urea can also assist with this process and may be effective together with sonication (high-powered, not a bath sonicator). There are also detergents that are sold for mass spec work that can be precipitated out after lysis that may be helpful. A question back to you is why you are looking to avoid sample clean-up (SPE)? Perhaps you just need an alternative clean-up product.
Thank you for your responses.. Dr Ottens, it is true, that without RIPA we will not get to membrane proteins as easily as sonication alone? Regarding the SPE columns we do not want to avoid them, I just wanted to know if the sonification can replace completely RIPA buffer .. It would be more comfortable .. and I was a little concerned about the potential loss of protein after application of SPE colums .. But it will be probably without loss. .. Do you have any used protocol to use them? Thank you!
Miroslav Ferko, perhaps you could describe what exactly are your concerns about using RIPA buffer?
It seems you will need to use detergents or something else that disrupts membranes. The reminder of the buffer composition according to the listing by Engelbert Buxbaum seems to be rather innocuous.
Miroslav, with detergent buffers (like RIPA) I generally clean my lysate with the Pierce Detergent Removal Spin Column resin. I have found that this is very effective at cleaning up determine (and urea too) from protein samples, and can work well with small and larger volume samples. I buy the larger 4mL columns (Thermo Pierce Cat No. 87779), resuspend the packing and then pipette what I need into a smaller 500 uL centrifuge cartridge. E.g., to work with 20 uL of sample, I'd pipette 125 uL of Detergent Removal slurry into the 500 uL support, then wash with 400 uL of a simple buffer like TBS, then add the sample and incubate (see product manual) and then elute protein off the column with 25 uL of TBS. The advantages are flexibility with sample volume and no need to use organics that could cause insolubility issues. The eluted sample can then go straight onto preparation for mass spec.
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