contamination in protein expression - I got a couple of questions (Dec/14/2010 )

Hi all, I need your professional advices since I do not have any senior or technician to guide me in my place:

I had cloned and expressed 4 different his-tagged proteins with E.coli BL21(DE3). For some reason, i had to get the native form of my protein. For the protein purification I use Protino Ni-TED 1000 (Marcherey-Nagel). I had collected the cells from whole broth of 200ml and re-suspend into Lysis-Equilibration-Wash buffer (LEW buffer:50 mM NaH2PO4,300 mM NaCl, pH 8.0) in 20ml. (although manufacturer's recommendation is 5ml, I unable to do so because the sonicator in my dept only able to sonicate 20ml and above), Add lysozyme to a final concentration of 1 mg/ml )for native protein purification), rocker in room temp for 30 minutes (manufacturer: stir on ice), followed by sonication 10 x 15 s bursts, centrifuge to remove debris and filtered through a filter membrane. The rest is just followed the protocols for the binding and elution, and I run through the column with all the ~20ml sonicated protein sample I have, since there is no mention of the volume of sample I can load into the column.

After the 3 elution, I pooled all it together ~5ml and done a buffer exchange (into TBS buffer) in a vivaspin column, and concentrated it till ~80ul. My protein's concentration here is ~8mg/ml, rough estimation by BCA method.

I had done a PAGE gel analysis (NuPAGE, MES) and western-blotting (alkaline phosphatase on PVDF). Attached is my gel picture.
The red box indicated the estimated size of the protein I suppose to get,~12kDa . ABCD were my protein lanes, L is the protein ladder (prestain ladder from invitrogen)

My question is:
1) in my lane A: is it meaning to say that my protein was degraded or too concentrated?
2) did I overload too much protein into my gel?
3) any suggestions to greatly reduce/total eliminate the unwanted proteins?
4) did I over silver stain my gel and western blot?
5) there is a smoke-like appearance in the middle of my WB, any explanation?
6) any other suggestions and comments are welcome

Thank you very much for your kind help. I can't find guide/help in my dept and I really depend on you guys.
Million Thanks.

adrian kohsf on Tue Dec 14 21:48:25 2010 said:

too concentrated.

yes

purify your protein of interest to totally eliminate. you can try adding 0.1% tween-20 to your wash buffer and antibody solutions, also add blocking agent to antibody solutions. dilute antibodies more.

staining looks good to me

one explanation is handling during staining procedure (eg- pouring solutions directly onto membrane, membrane sticking to staining vessel)

Hi mdfenko,
Many thanks...

I completely agree with mdfenco. If you would load 10 times less the gel, your proteins would look like much purer, as you are overloading the gel, any little contamination seems to be higher.