Have you got that down to an art yet as I have a few questions if I may from an extraction meister so to speak:
It's an art easily mastered. Sounds like you're well on track!
So if I separate the membrane / soluble proteins using Prot-Two Kit from Sigma. My final samples are dissolved in 40 mM Tris, 7M urea, 2M thiourea and 1% C7BzO detergent, pH 10.4 (and they have already been treated with TBP/IAA). Do you think that these extracts are compatible with SDS-PAGE/Western blotting?
You're clearly thinking too much, that's only relevant in a lab for testing purposes! After extracting the urea you simply filter it through a negatively charged gauze and extract the free radicals! You will notice that the filtrate smells like ****
First, I was wondering about pH - I'm afraid if I mix those samples (pH 10.4) with my 6X sample buffer, the final pH value will hardly be 6.8, won't be? Is correct pH of sample critical for running and protein resolving?
pH has little effect on power! Proton only really affects flavour! High pH, don't gargle
Second, is it possible to heat those urea-based buffers as I do in 'standard' protocol to enhance protein denaturation (65C, 15 min)? In the product info, there is written that these buffers should not be heated above 30C to avoid formation of cyanates which could damage proteins.
Is the combination of urea/thiourea/detergent at above mentioned concentrations enough for effective protein denaturation (so there is no need to heat samples), or could be better denaturation achieved with heating in the presence of e.g. SDS?
Now you're taking the ****!! Heating is worth while in that it concentrates the urea and moves it up the ochre scale and helps to liberate more power
If I decide to precipitate proteins (or maybe ultrafiltration could do a good job as well?), what would you suggest for protein solubilization? Just 1x sample buffer for SDS-PAGE? (I don't need to measure protein concentrations, so bromophenol blue would not interfere)
Only to happy to provide a sample for you to practice with I'd recommend gargling to sample the protein concentrate! Filter first or there may be more free radicals (swimmers) than you bargained for
Any advice is greatly appreciated.....
If I had known it'd get this in depth I wouldn't have used my bloody iPhone!!!