DNA precipitation and not protein - specific.. need it now!! (Oct/26/2006 )
I have an unknown sample.. which may have protein or DNA or something else in it.. it has high 260 & 280 absorbance.. ratio almost 1. I need to identify what it is.
I wanna try precipitating using something that can precipitate either DNA or Protein, not both. Most alcohols and salts work on both i know.
Can anyone help pls?
do a phenol chloroform.
pick the upper phase and precipitate with IprOH or NaAcetate/100%EtOH.
Then carefully removes the aqueous phase on the tube1.
let say you have 0.5ml of phenolic phase.
Add 0.3ml 100%EtOH
Let sit RT 2-3'
Spin 2000g 10' 4° : that will pellet the remaing nucleic acids.
To supernatant : add 0.75ml IprOH
let sit 10' RT
spin 4500g 10' 4°
vortex the pellet with 1ml ethanol let sit 20' RT, spin again 4500g 10' 4°.
resuspend proteins in RIPA buffer
thanks fred.. i shal try that n tell u if it helped:)
anyone else knows how wud be able to identify whats the sample.. basically.. lemme elaborate.
i have this cell lysate (cell's r supposed to b producing a cytoplasmic soluble recombinant protein -nuclease). we use rp-hplc to verify production of the protein. now, what i m seeing is.. that when we change the fermentation scale for the growth and induction.. (i.e. instead of setting up a 3L fermentor the usual, we set up a 10L fermentor). on this change.. what we saw was a strange impurity popping up in the cell lysate. this impurity is present starting the 4th feed (whatever.. i mean.. an early stage) before induction.. and increases over time. on induction our protein increases majorly.. impurity increases li'l.
the impurity gives a peak on rp-hplc.. ratio of 260/280 for this is ~1. while tht for the protein is ~0.5.
between the above different stage.. fermentation samples.. the sds-page does not show any major difference in the profile except for the obvious increase in the protein of interest on induction.
when collected (the peak for the impurity) and run on an sds-page.. there's absolutely no protein band. (i've made sure nothing's going wrong in the experiment.. like gel%, loading amt etc.. as much as i cud;) and have confirmed the results).
so.. now i suspect it cud b dna or lipid or some byproduct or something.
now, can anyone help me with this?
i plan on:
1. fred's pptn strategy.
2. running a dna-etbr gel
3. dong some biochemical test for dna/lipid.
pleeeeeeease tell me how else i can identify this one..!!
thanks a ton!