Transient transfection with large plasmid - (Feb/22/2014 )

Hi, 

I cloned my GOI into a vector to add an N-terminal Flag tag and I am trying to verify expression after transient transfection by western blot with an anti-Flag M2 antibody (mouse mono) at 1:1000 dilution and 20ug total protein. My western does not show any any bands even after long exposures (10min). A major issue is that I do not have a positive control but I plan to get one. This construct is 10kb and GOI is 5kb. I have never transfected with such a large construct. Do I need to use more plasmid DNA compared to a construct that is 7-8kb? Either the transfection or western (i.e. antibody) is not working. I also tried a rabbit polyclonal anti-flag antibody at 1:1000 but got lots of bands. I do not care about detecting the mRNA in this case. I am trying to confirm expression of the Flag tagged protein. 

Any suggestions?

Note: I transfected the same cell line exactly the same way but with another expression construct 7kb (HA tagged GOI - 2kb) and I was able to detect this protein with an anti-HA antibody. Ponceau S stain and actin ok for both.   

Thanks!

It should work fine with the same methods as you use for smaller plasmids.  The issue with amount of DNA means that there will be fewer copies present per ug than there would be for a smaller plasmid, however if the promoter is a viral one, this shouldn't be much of a problem with detection.

I take it you have tried all the different DNA amounts and ratio things that are usually used to optimize these sorts of things?  Could you try to load more protein onto the gel?  10 min is not a long exposure - try 1/2 hour or more.

Do you have a known flag +ve lysate you could use to test the antibodies?  That's the only real way to be sure that it is or isn't an antibody problem.

In the past I have had problems expressing single flag tags, which was probably due to folding problems, where the flag ended up folded into the protein and wasn't available for the antibody.

bob1 on Sun Feb 23 19:32:11 2014 said:

It should work fine with the same methods as you use for smaller plasmids.  The issue with amount of DNA means that there will be fewer copies present per ug than there would be for a smaller plasmid, however if the promoter is a viral one, this shouldn't be much of a problem with detection.

 

I take it you have tried all the different DNA amounts and ratio things that are usually used to optimize these sorts of things?  Could you try to load more protein onto the gel?  10 min is not a long exposure - try 1/2 hour or more.

 

Do you have a known flag +ve lysate you could use to test the antibodies?  That's the only real way to be sure that it is or isn't an antibody problem.

 

In the past I have had problems expressing single flag tags, which was probably due to folding problems, where the flag ended up folded into the protein and wasn't available for the antibody.

First thing I will follow-up with is the positive control. Trying to find a lab with a positive lysate or a Flag construct. I have tried different DNA and ratio amounts in the past but with the a different expression vector. I could load more protein but I assumed the protein would be easily detected at 20ug because the exogenous expression is driven by a CMV promoter.  I have read about the issues with a 1X flag tag. I assume that is why people are using things like a 3X. I chose the 1X to make the tag as small as possible because I did not want it to interfere with the protein function. I may consider a 1X vs 3X Flag tag if the positive control works and I cannot detect my sample even after loading more protein. I know the sequence is correct as I have sequenced the entire 5kb insert. 

Thanks!

Epigeneticist on Sun Feb 23 23:04:50 2014 said:

First thing I will follow-up with is the positive control. Trying to find a lab with a positive lysate or a Flag construct. I have tried different DNA and ratio amounts in the past but with the a different expression vector. I could load more protein but I assumed the protein would be easily detected at 20ug because the exogenous expression is driven by a CMV promoter.  I have read about the issues with a 1X flag tag. I assume that is why people are using things like a 3X. I chose the 1X to make the tag as small as possible because I did not want it to interfere with the protein function. I may consider a 1X vs 3X Flag tag if the positive control works and I cannot detect my sample even after loading more protein. I know the sequence is correct as I have sequenced the entire 5kb insert. 

 

Thanks!

I have found that the ratio thing needs to be optimized for each vector, but it is relatively rare to have one work better at anything other than a 3:1 ratio. 

My issues were with 1x flag, which I was using for the same reasons as you.  The signal was there, it was just very weak, and my protein wasn't expressing well at all due to some toxicity effects.