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Which Tag is most suitable


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8 replies to this topic

#1 Purzel

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Posted 19 May 2010 - 09:58 AM

Dear all,

I would like to clone a gene. And after all cloning steps I would like to transfect cells. According to the literature my protein of interest is shuttling between cytosol and nucleus. Now my question. Do I have to care about the tag. All common tags (e.g. His, FLAG etc.) will not hinder the shuttling? Which vector is suitable for this experiment?
All comments are welcome.

Kind regards,
Purzel

#2 bob1

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Posted 19 May 2010 - 05:34 PM

Do you need to tag the protein? If you are overexpressing it, as you will be with a plasmid, you should be able to detect level changes in the cell via immunofluorescence or nuclear fractionation and western blotting.

Tags may or may not interfere with the normal function of the protein and/or its cellular distribution. If you do tag it you would be advised to do both an N-terminal and a C-terminal tag (separately) and test for distribution/ behaviour changes. You would also be advised to use as small a tag as possible, things like GFP are big and probably will interfere in some fashion.

#3 Purzel

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Posted 20 May 2010 - 03:22 AM

Do you need to tag the protein? If you are overexpressing it, as you will be with a plasmid, you should be able to detect level changes in the cell via immunofluorescence or nuclear fractionation and western blotting.

Tags may or may not interfere with the normal function of the protein and/or its cellular distribution. If you do tag it you would be advised to do both an N-terminal and a C-terminal tag (separately) and test for distribution/ behaviour changes. You would also be advised to use as small a tag as possible, things like GFP are big and probably will interfere in some fashion.



Dear Bob1
Thanks for your reply.
Could you answer another question. Is it always the case that I should see a difference in the expression level after expressing the protein. I mean I transiently transfected my cells with a wildtype plasmid and neg dominant mutant plasmid. I could verify the successful transfection by detecting the tag of the plasmids. After this I have checked the protein and I saw the same expression level in both samples. Or is this an exception.
Thanks a lot
Purzel

#4 bob1

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Posted 20 May 2010 - 05:00 PM

You did untransfected controls right? If so, you should be able to see a higher level of protein in the transfected vs untransfected cells with most plasmids. Most (but not all) plasmids for eukaryote expression run viral promoters which give very high expression levels. Check that the plasmid is correct by sequencing.

#5 laurequillo

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Posted 20 May 2010 - 09:56 PM

Do you need to tag the protein? If you are overexpressing it, as you will be with a plasmid, you should be able to detect level changes in the cell via immunofluorescence or nuclear fractionation and western blotting.

Tags may or may not interfere with the normal function of the protein and/or its cellular distribution. If you do tag it you would be advised to do both an N-terminal and a C-terminal tag (separately) and test for distribution/ behaviour changes. You would also be advised to use as small a tag as possible, things like GFP are big and probably will interfere in some fashion.

Bob1 is right, be careful with the position of the tag. The his tag is useful for some things (if you want to do NiNta purifications...) but it is too inespecific and not good to work in IF for example. You should try first Flag tag, or gfp (for IF)
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#6 Purzel

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Posted 21 May 2010 - 02:49 AM

You did untransfected controls right? If so, you should be able to see a higher level of protein in the transfected vs untransfected cells with most plasmids. Most (but not all) plasmids for eukaryote expression run viral promoters which give very high expression levels. Check that the plasmid is correct by sequencing.



Thanks Bob and Laurequillo,

I would like to come back to the expression level. After Ponceau staining of my membrane should I see an enhanced band in my transfected samples compared to the non-transfected cells? Or do I have to wait for the ab detection. From your experiences would you check after 24 h oder earlier?

I sequenced my plasmids and they were okay.
Thanks a lot
Purzel

#7 eurisko

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Posted 21 May 2010 - 07:12 AM

Dear all,

I would like to clone a gene. And after all cloning steps I would like to transfect cells. According to the literature my protein of interest is shuttling between cytosol and nucleus. Now my question. Do I have to care about the tag. All common tags (e.g. His, FLAG etc.) will not hinder the shuttling? Which vector is suitable for this experiment?
All comments are welcome.

Kind regards,
Purzel


i have cloned a gene in a pcDNA3.1+ and tagged it with the HA tag. this worked well for me. my protien shows up well on my westerns, immunofloresence and flow cytometry using the anti HA antibody.
all the best

Edited by eurisko, 21 May 2010 - 07:23 AM.


#8 rkay447

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Posted 21 May 2010 - 08:49 AM

You may not be able to see an enhanced band in Ponceau. Ponceau just isn't that sensitive and only shows you the most abundant proteins which may not include your protein, even when overexpressed. Sometimes I can see it, sometimes I can't. The western blot is much more definitive. You should always run one lane of untransfected lysate to compare with the transfected. This way you can compare the expression levels if using an antibody to the specific protein and you can determine what bands are "background" if using an antibody to the tag. The tag you want to use depends on the experiments you want to carry out. As others have mentioned, the GFP or RFP are great for IF (and essential for live cell imaging) but are very large and can certainly disrupt protein function, localization, interactions, ect...so I try not to use these. My favorite tag is HA. It is very small and works wonderfully in IF, western, IP, flow. I only use one HA tag so my IPs tend to be a little weak so if I know I'm going to do an IP I'll go ahead and add 6 HA tags but the one tag makes for incredibly clean IF photos. Some people in my lab are fans of the flag tag but I've never had great success with this one except for westerns. My other "go-to" tag is Myc. Again, it's very small and works very well in all assays I've done.

#9 Purzel

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Posted 22 May 2010 - 05:05 AM

You may not be able to see an enhanced band in Ponceau. Ponceau just isn't that sensitive and only shows you the most abundant proteins which may not include your protein, even when overexpressed. Sometimes I can see it, sometimes I can't. The western blot is much more definitive. You should always run one lane of untransfected lysate to compare with the transfected. This way you can compare the expression levels if using an antibody to the specific protein and you can determine what bands are "background" if using an antibody to the tag. The tag you want to use depends on the experiments you want to carry out. As others have mentioned, the GFP or RFP are great for IF (and essential for live cell imaging) but are very large and can certainly disrupt protein function, localization, interactions, ect...so I try not to use these. My favorite tag is HA. It is very small and works wonderfully in IF, western, IP, flow. I only use one HA tag so my IPs tend to be a little weak so if I know I'm going to do an IP I'll go ahead and add 6 HA tags but the one tag makes for incredibly clean IF photos. Some people in my lab are fans of the flag tag but I've never had great success with this one except for westerns. My other "go-to" tag is Myc. Again, it's very small and works very well in all assays I've done.


Hi rkay447,
thanks a lot for your explanation.
In the first reply Bob suggested that I should generate both C-terminal and N-terminal constructs. What it is your opinion. Might the position of the tag destroy the function of my protein? Or it doest matter.
Cheers
Purzel




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