Running bacterias as control on SDS-PAGE - should I see anything without breaking them? (Feb/15/2010 )
My thesis problems continue 
My goal is to generate an antiserum against human protein (c-terminal part). First I designed primers and cloned the desired insert. It has been sequenced, everything was fine and I have two histidine tags. I have used BugBuster for protein extraction and my desired band is in inclusion bodies (Coomassie Blue staining). I have also proved that I have at least one histidine tag by doing Western blotting (which I finally got working after changing the entire protocol, but it's working and I get one clear band without background, so I take it ). My next step would be to do silver staining... but my lovely boss also wants me to run bacteria samples in a gel as according him I should see clear band on my positive control compared to the negative sample. He is telling me just to grow them and run them on the gel...
but, I feel I'm sort of stupid, but without breaking the bacterias, can I actually see anything by just running them on the gel? I did something like this once before and saw nothing... and someone said that the problem was that I haven't even broken the bacterias and released the proteins (and the desired protein is still in inclusion bodies)... so, if I do what my boss tells me to do and see nothing, is the problem my protein or the protocol? Needless to say I'm not going to do my Ph.D. in this place... my supervisor knows things as well as me and can't help with anything... according him this is how I should prove that the protein actually exists and yet I have found zero experiment like this online
Juliasarmoire on Feb 15 2010, 06:13 AM said:
My goal is to generate an antiserum against human protein (c-terminal part). First I designed primers and cloned the desired insert. It has been sequenced, everything was fine and I have two histidine tags. I have used BugBuster for protein extraction and my desired band is in inclusion bodies (Coomassie Blue staining). I have also proved that I have at least one histidine tag by doing Western blotting (which I finally got working after changing the entire protocol, but it's working and I get one clear band without background, so I take it ). My next step would be to do silver staining... but my lovely boss also wants me to run bacteria samples in a gel as according him I should see clear band on my positive control compared to the negative sample. He is telling me just to grow them and run them on the gel...but, I feel I'm sort of stupid, but without breaking the bacterias, can I actually see anything by just running them on the gel? I did something like this once before and saw nothing... and someone said that the problem was that I haven't even broken the bacterias and released the proteins (and the desired protein is still in inclusion bodies)... so, if I do what my boss tells me to do and see nothing, is the problem my protein or the protocol? Needless to say I'm not going to do my Ph.D. in this place... my supervisor knows things as well as me and can't help with anything... according him this is how I should prove that the protein actually exists and yet I have found zero experiment like this online
You can boil a bacterial pellet in sample buffer and run on a gel for as a whole cell lysate. You will just need to play around with how many bacteria to pellet before the sample is too concentrated. If it's too concentrated, it will run poorly on the gel, and may affect your western results. Of course, you will also need an internal control to show that the positive and negative samples are loaded equally.
That's what I do:
10 uL of bacterial culture
5 uL of Laemmli buffer (don't forget b-mercapto)
Mix, boil for 5 minutes and load on gel.
Simple. That's what I always see:
Juliasarmoire on Feb 15 2010, 09:13 AM said:
Laemmli buffer contains SDS and the boiling steps WILL disrupt cell walls!