It's a tricky question!

Are your cells differentiation related to protease inhibitors? If you have some inhibitors that do not interfere with the differentiation, perhaps one could be used to prevent the degradation of your protein.

If you are getting a clean cleavage product maybe look into the sequence at which this event occurs. Can you transfect a tagged version (that localizes properely) of this protein and purify it? Maybe you can come up with a stable point mutant that still behaves naturally?

Use of protease inhibitors may be the way to go but they are usually short-lived at room temperature. You may consider using protease inhibitors at different time points during your culture.

Hi!

I agree with Shujuan Gao. It can be that the degradation IS PART of the role of the protein during differentiation. I would recommend you to isolate the protein, sequence the "degradation" product and look what domains it has. Since after 48 hours you don't see this degradation, I doubt that it is a product of overexpression.

Good luck,

Luisa

this is a relatively long period for transient transfections, it could be that the protein is just been poorly/ improperly expressed in the cells at this time point. if you are getting appropriate expression at 48 hours then not at say 6 days then it may just be to do with the transient nature of your transfections. Typically expression goes up within the first 48 and then falls dramatically. when we do it for a membrane protein we also find that at later time points (with certain techniques) the proteins appear internalized and no longer at the membrane? you could consider other techniques such as nucleofection (Lonza) that can produce longer lasting effects. Sometimes slowing down cell cycling rate can reduce these effects too (slow down cell cycle, low serum medias - but then that could affect your differentiation process? or you could try get a stable transfection but again differentiation might be harder to measure in those cells

This is an interesting observation. You maybe onto something. I agree with some of the previous answers: there is a possibility that this cleavage is a specific natural process regulated by cell confluency or differentiation stage. Certainly, it may also just be non-specific degradation. Is the 50Kd consistent with the length of TM-Cytotail or that of the extracellular domain of the protein? The cleavage may occur extracellularly, endosomally, cytoplasmically, or non-specifically during cell Lysis. You can apply hot (100C) SDS-PAGE sample buffer directly to cells and shred DNA with fine needles to avoid protein degradation during cell lysis.

--If you still see 50kD fragement, I agree with other answers that you may want to determine the cleavage site by N-terminal sequencing of the fragment. You can also do a time course (2day, 3day, 4day, 5day and 6day) to determine when the cleavage occurs, see if that correlates with any other observations (cell density, morphology, differentiation, etc.). Anyway, you get yourself an interesting project to work on.

--If the protein stays at 135Kd, you got a non-specific degradation problem. I have seen seen similar problem with a chloroplast envelope protein Toc159, which was initially identified as a 86kD protein due to degradation during preparation of chloroplasts. We were able to preserved the full-length protein by adding Sigma's protease cocktail in all buffers.

In addition, you could also try change cell media frequently with protease inhibitor containing fresh media to see if it prevents degradation during cell culture.

Hi, Genevieve, if aim of your experiment is to test effect of the your protein on cell differentiation, you should use some differentiation marker. May be you should test how rapid transfected and control cell undergo differentiation? May be one of the marker you can use is EdU as a marker of DNA replications. If cell go to differentiation, they will loose DNA replication ability. Just compare control cell and transfected one. May be you can also use other differentiation marker. I think, in such way you will have a direct answer on your question.

By addition of extra protease inhibitor you will change degradation of so many proteins. It will be difficult t conclude about the effects of transfected protein itself

Good luck!

I agree with Xuejun Chen. A time course western blot for your protein would provide answer to your question. In addition to blotting protein of your interest, you should also probe for any known differentitaion marker in your experimental cell lysate as well as in control cells undergoing differentiation.

Good luck.