Starting to express protein - tips needed to start it =) (Jan/09/2007 )
My expression failed miserably. Hehe... no desired protein was spotted! ARGH!!!
But I am pretty alright... cause lucky enough to spot a rainbow when I was on my way back home. 
Anyone got problem with expressing protein using the HisTag system? Stupid HISTAG! maybe should use the GATEWAY technology.
I have another question in my mind. One of my protocols require MSE Sonicator with the rod for lysing the cells. In my lab only have the normal water sonicator. So is it ok if I use that to lyse my cells? But then the amplitude of the water sonicator is much lower than the rod one. So I think it is not sufficient enough to lyse the cells. Any idea?
Any other idea to lyse the cells? How about just heating them up (that's what recommended in Sambrook)
Thanks guys and girls 
I got two different bands from SDS. One at 25kDa and the other one is 50kDa. Do you guys think it will be dimer formation? But i thought since it is denaturing method, so all the protein will be denatured and just become the monomer?
Should I increase the heating time to break the dimer? Or should I allow longer incubation during the lysis part?
Where is the queen of expression? 
Thanks for reading and all replies will be more than being appreciated.
Should I increase the heating time to break the dimer? Or should I allow longer incubation during the lysis part?
Where is the queen of expression?
Thanks for reading and all replies will be more than being appreciated.
you should increase the heating time, but be careful that you don't heat too long at boiling temperature. you can heat longer at lower temperature (80C, 60C).
hmm thanks mdfenko!
I am using urea in my SDS loading buffer, so I can't really heat up my sample once added. So I have to heat up before adding the loading buffer.
I will try today and see how it goes. Thanks alot.
I am using urea in my SDS loading buffer, so I can't really heat up my sample once added. So I have to heat up before adding the loading buffer.
I will try today and see how it goes. Thanks alot.
why are you using urea? you will denature all your protein! you shouldnt use urea! 2 bands? can it be that your protein is degraded? so at first you didnt get any protein and then you got 2 bands right? what did you change? please tell us more details.
we also have water sonicator in our lab. it gave me comparable results to the lysozyme. by the way you can do both sonication and lysozyme.
Well, i used urea to get denatured protein for initial screeening only. Not for any further characterisation yet.
I changed the method of lysis. I used alkaline lysis together with urea this time to lyse the cells.
Perhaps I will try out the water sonicator. Hope it is strong enough to lyse the cells.
TQ!!!
what do you mean? It is in my lysis buffer, to solubilise protein. If using guanidine hydrochloride, it wil solubilise hydrophobic protein.
I didn't use any physical lysing method. Did i make any mistake? 
timjim, i still cant understand why are you using Urea? it will denature your protein. you will have to renature it after that. I mean if your protein can be solubilized with milder solvents, dont use URea. save it till the end.