problem dissolving protein pellet after aceton precipitation - (Oct/14/2008 )
Hi forumreaders,
i hope you can shed some light on my problem because it is starting to be a time drain on my work now.
After strep purification i elute my proteins in 400µl elution buffer (300mM KCl, 40mM Tris-HCl pH7.4, 5mM beta-mercaptoethanol, 2mM EDTA, 0.1% NP40, 2.5mM biotin). After bradford reading i have about 15µg protein in these 400µl so i would like to precipitate the proteins to load them on an SDS page. Then the mystery starts.
- after acetonprecipitation i get a bright white pellet which doesn't dissolve at all in SDS sample buffer + 8M urea. The only thing that helps is heating it for 30 seconds at 70°C but it just precipitates again after 2 minutes of RT. So basically i can't load it on a gel (i tried and i get crystals in the slot).
--> i tried : heating it for 1h at 50°C (after sample cools down it precipitates again), taking out the urea from the sample buffer (no change), boiling (only temporairy fix until sample cools down)
BUT:
- testing the same conditions (same buffer, O/N acetonprecipitation, 30mins drying RT) with BSA or any celysate doesn't deliver this bright white pellet. It is more a transparent pellet then a white one and it dissolves very easily in 20µl SDS sample buffer.
I would really like to find out what is going on and how to fix it and i hope one of you has had this before or knows what to do. Is it better to use TCA with this buffer? Is there any way to save my sample (now in 400!!µl of SDS sample buffer)?
Thanx
kitty
i hope you can shed some light on my problem because it is starting to be a time drain on my work now.
After strep purification i elute my proteins in 400µl elution buffer (300mM KCl, 40mM Tris-HCl pH7.4, 5mM beta-mercaptoethanol, 2mM EDTA, 0.1% NP40, 2.5mM biotin). After bradford reading i have about 15µg protein in these 400µl so i would like to precipitate the proteins to load them on an SDS page. Then the mystery starts.
- after acetonprecipitation i get a bright white pellet which doesn't dissolve at all in SDS sample buffer + 8M urea. The only thing that helps is heating it for 30 seconds at 70°C but it just precipitates again after 2 minutes of RT. So basically i can't load it on a gel (i tried and i get crystals in the slot).
--> i tried : heating it for 1h at 50°C (after sample cools down it precipitates again), taking out the urea from the sample buffer (no change), boiling (only temporairy fix until sample cools down)
BUT:
- testing the same conditions (same buffer, O/N acetonprecipitation, 30mins drying RT) with BSA or any celysate doesn't deliver this bright white pellet. It is more a transparent pellet then a white one and it dissolves very easily in 20µl SDS sample buffer.
I would really like to find out what is going on and how to fix it and i hope one of you has had this before or knows what to do. Is it better to use TCA with this buffer? Is there any way to save my sample (now in 400!!µl of SDS sample buffer)?
Thanx
kitty
have you tried dissolving it in the same elution buffer? let's say you have acetonprecipitated your protein, and removed aceton and supernatant. then add like 50-100 ul of the same elution buffer and use 6x SDS-PAGE buffer not to dilute your protein more....sometimes people use 2x SDS-PAGE buffer...this would dilute your sample 1/2....have you tried this?...also any possibility that the nature of your protein is insoluble? even in your first eluted sample?
i haven't tried that but after many test experiments i assume that it is indeed the nature of my protein...unfortunately.
there will remain some protein undissolved mainly cytoskeleton proteins; SDS sample buffer should dissolve most of it...