Hi!
I am trying to express my protein of interest in a TNT cell free expression system (Promega, L1170). My protein is tagged With V5 and His tags. The problem is when I run the samples in a western blot, though I can see a band of the proper molecular weight (about 37 kDa), I see a smear under it. I attach a picture so you can see what I mean. In the lines 2 and 3 I am using the anti HA ab, and in 4 and 5 the anti His. (The first one is a marker and in the last two I am using an antibody against the protein that does not seem to recognize my tagged protein very well).
I followed the instructions of the manufacturer. I also tried adding protease inhibitors, but I get the same results.
Anybody has any ideas?
Your protein might run at the same MW as a very abundant protein in the TNT Lysate and might get pressed away in the gel and during transfer. Or it could be unspecific binding of the antibody due to the high amount of a protein there.
I would load the next time on the same gel the same amount of "unprimed" TNT to check by Ponceau S whether there is an abundant protein running at the same MW. At the same time this will give you the background signal of the TNT in the western blot.
Hope this helps.
Thanks! I don't think it is unspecific binding, because I have also tried previously to load the same ammount of a similar TNT reaction in which I have not added the plasmid, and also I have tried using other plasmid. In both cases I do not see any unspecific binding of my antibody for that molecular weight, and no background, it looks very clean.
In the blot I Attached on the question I have loaded 5 ul of my reaction, I have tried loading 1 ul, and in that case it looks more like a double band. Here Attached this blot, the 1st lane contains the TNT reaction, probed with the V5 antibody. The last one is the marker.
Do you think it can be then a matter of amount of material? or "artifact" during the running/transfer?. For this blots I used a 10% gel, and my protein should be around 37 kDa.
Hi Kelly, indeed with the unprimed TNT and the anti-V5 it looks nice.
Hmm, it could be the amount of material (is there a highly abundant protein running at the same size?) or it could also be that your protein is modified in the RRL?
http://www.promega.com/~/media/files/resources/product%20guides/in%20vitro%20protein%20expression/ivex_chp8.pdf?la=en
Eventually running a 12% Gel would help in the case that another high abundant protein runs at the same size and presses the protein away. Maybe a 12% gel in combination with long running would separate it further. However, since you say that when you load less it looks like a double band this might indicate that your protein might get modified or processed in the RRL. Is anything known whether this protein is normally modified or processed?
The double band may also indicate that you have an internal methionine that serve as a start point for translation.
This could only work as an explanation if the both your antibodies give double bands ad your tags are C-terminal fusions.
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