I'm using a protocol that has been in my lab for years without anyone having issues previously. We extract protein from parotid glands (salivary glands) of mice- I use C57BL/6J mice, while others in my lab use primarily FVB mice.

The protocol is as follows:

Samples are snap-frozen on dry ice following dissection and the protein extraction is done with frozen tissue. Samples are kept on ice through the entire extraction protocol.

Make a master mix of lysis buffer: RIPA buffer (500ul/sample; made in the lab), Protease inhibitor cocktail (7.5ul/sample; Sigma: P8340), Sodium orthovanadate (25ul/sample of 100mM stock; made in the lab), Sodium fluoride (5ul/sample of 100mM stock; made in the lab).

Add 500ul of master mix to each sample and homogenize with a Dounce homogenizer in a tube on ice.

Add 3ul PMSF (100mM stock; made in the lab) following homogenization.

Sonicate samples on ice with 10s intervals sonicate/rest (or less time sonicating if sample begins to foam). Sonicate for a total of 2 minutes.

Centrifuge samples at 12,000 RPR at 4 degrees C for 10 minutes.

Collect the supernatant in a new tube and determine protein concentration via Bradford assay.

Initially, my protein concentrations look "normal" (10-20ug), but following a single freeze/thaw cycle they degrade by 50% or greater, with some as low as 0.5ug.

I have tried increasing the concentration of protease/phosphatase inhibitors (with many different combinations) and increasing the volume of RIPA buffer per sample (to 750 or 1000ul) but my samples still end up degraded by 50% or greater by the next freeze/thaw cycle (measured by Bradford). I went to another lab that extracts protein from multiple mouse organs (liver, kidney, spleen, intestine, etc.) and tried their lysis buffer/PI cocktail mix and had the same problem. I am unable to use many of my samples because the concentration of protein is too low and I can't seem to figure out what is causing this.

Others in my lab follow the same protocol and don't have this problem so it doesn't seem to be an issue with the buffers or inhibitors.

Does anyone have any pointers on things to try? I appreciate all pieces of advice!!