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Overexpression plasmid is not expressing protein


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13 replies to this topic

#1 gingernut_7

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Posted 01 April 2011 - 12:48 PM

Hi,

I am trying to over express a demethylase protein in a lung cancer cell line but my cells are refusing to express the protein.

I know the transfection is good because I have co-transfected my CMV-KDM (lysine demethylase) plasmid and CMV-GFP into the lung cancer cell line. The GFP was detectable by flow cytometry and western blot, therefore the cells were transfected.
I know the plasmid is good because I have had it sequenced and it also over expresses at an mRNA level, validated by qPCR, in my lung cancer cell line.
I have also overexpressed this plasmid in HeLa and probed for anti-flag and detected the protein in HeLa.

So the lack of protein expression is cell specific. So far I have transfected HeLa which do express the protein, and 2 lung cell lines which do not express the protein.

I am super excited that it might be a lung cancer phenomenon. I am looking at other cell types to make sure first.
But I have no idea where to go from here. There are many possibilities of mechanisms that can be inhibiting translation (the mRNA is expressed but the protein is not).
Can you please make suggestions of what and how I can look at possible translational inhibitors.

Much appreciated.

#2 Rsm

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Posted 02 April 2011 - 11:14 AM

Not so many translational inhibitors that are specific for a given mRNA... Are there any miRNA that target this mRNA?
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#3 Ambinlab

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Posted 19 April 2011 - 10:29 AM

Hi,

I am facing the similar problem. I am trying to overexpress ING protein in HEK293s. I used 1ug GFP and 2.5 ug of my plasmid for transfection using lipofectamine 2000. GFP is detectable under fluorescent microscope. However, I did blotting and there was no change in bands from different samples.

Should I try different amount of dna?

Any ideas is much appreciated

Bests

#4 chimpsarehungry

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Posted 19 April 2011 - 10:34 AM

Hi Dreamer,

2.5 ug is enough for me to overexpress proteins in 293 cells. Are the GFP and your other plasmid using the same promoter?

#5 Ambinlab

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Posted 19 April 2011 - 10:39 AM

Hi chimpsarehungry,

I checked that, it seems that these two have the same promoter CMV IE. So Does that mean these two cannot be transfected together?

#6 chimpsarehungry

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Posted 19 April 2011 - 12:33 PM

Hi,

Yes the CMV promoter definitely works in 293 cells. And I assume you can transfect two plasmids at once. I usually do 2ug on 200,000 cells and let grow to confluency in a 6 well dish. Have people overexpressed with this other construct before?

#7 Ambinlab

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Posted 19 April 2011 - 02:24 PM

Yes, they have overexpressed 293s before with the same plasmid that I have. But it seems that I cannot transfect them together with GFP. Should I try the transfection of this plasmid alone (without GFP)?

#8 chimpsarehungry

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Posted 19 April 2011 - 03:49 PM

Yeah try that out. Good luck.

#9 Ambinlab

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Posted 26 April 2011 - 10:01 AM

OK, I did that, got an additional band in the sample that was transfected only with my plasmid. Again my GFP sample and GFP,plasmid sample had no difference. Can anybody explain this to me?

I am probing against actin by now to confirm there is a difference in band intensity.

#10 Rsm

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Posted 27 April 2011 - 12:00 AM

OK, I did that, got an additional band in the sample that was transfected only with my plasmid. Again my GFP sample and GFP,plasmid sample had no difference. Can anybody explain this to me?

I am probing against actin by now to confirm there is a difference in band intensity.


Are you using western blot or detection of GFP? IMHO most anti-GFP antibodies are really bad, detecting something random, so you'll see no differences between samples.
I got soul, but I'm not a soldier

#11 Rsm

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Posted 27 April 2011 - 12:01 AM

OK, I did that, got an additional band in the sample that was transfected only with my plasmid. Again my GFP sample and GFP,plasmid sample had no difference. Can anybody explain this to me?

I am probing against actin by now to confirm there is a difference in band intensity.


Are you using western blot for detection of GFP? IMHO most anti-GFP antibodies are really bad, detecting something random, so you'll see no differences between samples.
I got soul, but I'm not a soldier

#12 Ambinlab

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Posted 27 April 2011 - 08:07 AM

I am not detecting GFP. My protein is not expressing if I co transfect it with GFP.

#13 genemall

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Posted 29 April 2013 - 01:18 PM

I know that this topic is 2 years old, but did you solve your problem? I have very similar situation and any help is wellcome.

#14 genemall

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Posted 29 April 2013 - 01:23 PM

Hi,

I am trying to over express a demethylase protein in a lung cancer cell line but my cells are refusing to express the protein.

I know the transfection is good because I have co-transfected my CMV-KDM (lysine demethylase) plasmid and CMV-GFP into the lung cancer cell line. The GFP was detectable by flow cytometry and western blot, therefore the cells were transfected.
I know the plasmid is good because I have had it sequenced and it also over expresses at an mRNA level, validated by qPCR, in my lung cancer cell line.
I have also overexpressed this plasmid in HeLa and probed for anti-flag and detected the protein in HeLa.

So the lack of protein expression is cell specific. So far I have transfected HeLa which do express the protein, and 2 lung cell lines which do not express the protein.

I am super excited that it might be a lung cancer phenomenon. I am looking at other cell types to make sure first.
But I have no idea where to go from here. There are many possibilities of mechanisms that can be inhibiting translation (the mRNA is expressed but the protein is not).
Can you please make suggestions of what and how I can look at possible translational inhibitors.

Much appreciated.




I know that this topic is 2 years old, but did you solve your problem? I have very similar situation and any help is wellcome.




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