No Land Rover in their drop down list....!!I think this is getting a bit off topic now
What I want to know, is if Blitz actually list the 200tdi in they're catalog!
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!!!
I think this is getting a bit off topic now
What I want to know, is if Blitz actually list the 200tdi in they're catalog!
Mmmmmmm tits 'n chocolate.......
Oh thats a shame, If Blitz did a dump valve for the 200 that would of swayed my decision to put that engine in an old rusty 2a I have kicking about. Now because of Blitz the poor old beast is destined for the scrappys becuase I will never have the power without that Tiiiiisshhhhhh
but words like that probably don't sink in and we can use them all day long to take the **** without you realising
Well maybe towards the end of the thread, I may have finally cottoned on (bit slow me) to the Urea Extraction - but my replies to the tuning question were valid.....Surely not all of them!
)
Well maybe towards the end of the thread, I may have finally cottoned on (bit slow me) to the Urea Extraction - but my replies to the tuning question were valid.....
Also so was the Scientific Query on the Ph Value of Protein extraction from Urea (Google is your friend sometimes
I pulled that out of thin air just to see who was listening I was quite impressed with the technicality of your answersWelcome To LandyZone, the Land Rover Forums!
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