do you sonicate or digest with lysozyme? - which one do you prefer and why.... (Mar/08/2006 )
wait a minute, Kathy
so, if I am getting this straight, you are sonicating, spinning, then running a portion of the pellet on a gel, while removing the supernatant to a column? and it is the spnt that is showing a big ol' sloppy smear? but your fractions contain very little protein?
before you worry too much about insolubility, I would worry about inefficient lysis
sounds like DNase might be a good idea? but I would definitely also add lysozyme too
thanx a lot everyone, Aimkins, i have done SDS-PAGE for all the fractions, i sonicate, centrifuge and then take samples of supernatant and the pellet...and the pellet has big amount of my protein (insoluble fraction) and the smear. supernatant had some protein (very little) which i went on to bind to the GST beads (sepharose glutathione)...washes had no protien and eluate has very very very little amount of protein...so that is why i am asuming that my protein was lost in the insoluble fraction, or in another words was not enough solubilized.... ....but i cant understand what is that smear??? ....thanx a lot for the advice.
at the step where you are seeing the smear, is it just the other proteins that live in the cells? you haven't separated them out until it goes on the column. you should see bunch of cellular proteins. it will look like a smear with some bands visible in it, with a big whopping band where your POI is supposed to be if induction was good.
would you please post a picture of what you are seeing?
If you didn't use DNase, it is possible that proteins stick on DNA, and then you lose your proteins wen you centrifuge and pellet insoluble fractions with DNA and bound proteins.
I never sonicate without adding DNase after sonication.
Did someone try?
How about the snap freeze method and just sonication, I am trying to get phosphoralated ERK1/2 map kinase. The paper i was trying to recreate used DTT in the sample buffer but I wanted a stock solution I can use for Western and protein assays. Do you think just sonication would be sufficient?
To obtain proteins to purify by chromatography steps I usually use sonication, and then I add streptomicyn, also you can use protamine, to the supernatant to eliminate nucleic acids. This method works well. Also I used glass beads and stirring, at 4 C, it's good and easy method.
Also I use sonication to obtain recombinant proteins, thaw and freezed is good, but in my personal opinion with sonication you can obtain more concentration of proteins.
thank you very much all for the information. this time im repeating with lysozyme, sonication and DNase, TritonX-100....(forgot to add DTT) ...aimkins, i have attached the results: first lane marker, second crude extract, third soluble, forth insoluble....and other are washes and etc....as you can see there is a smear in the insoluble portion and protein bands....(fat bands) thanx a lot for the advise
ive repeated again the experiment.....my sequence of events was: lysozyme 1mg/ml, on ice for 30min, add 0.2% TritonX-100 (same volume), shake tube vigourously, sonicate, DNase 30U/ml, centrifuge at 3000G for 30min at 4C. Seperate, bind to beads, wash and eluate. When I ran the gel it is almost the same as the one ive posted... i tried also without sonication, same results. What would you suggest??? is my protein terribly insoluble?? should I incubate with lysozyme in TritonX??? thank you for any suggestion....
no we dont have it here....
your lane from the insoluble fraction is overloaded. that, in itself can cause streaking. also, the insoluble fraction may need longer incubation with loading buffer prior to running the gel.