Fluorescence Localization for RFP Fusion but not GFP Fusion?

Use for discussion of various microscopy and imaging techniques, such as phase, fluorescence and confocal microscopy, electron microscopy, and various other imaging techniques.

Moderator: r.rosati

Fluorescence Localization for RFP Fusion but not GFP Fusion?

Postby klone » Sep 28 2012 8:08 pm

Hello Scientists.

I am doing some fluorescence localization of proteins using RFP and GFP tags in my bacterial species of interest.

I have tagged the same gene product with both RFP and GFP in separate recombinant bacterial strains. However, when I attempt to image these strains, I only get good signal/expected localization for the RFP tagged protein and not the GFP tagged variant. I get occasional localization with GFP tagged protein, but only in cells in which the tagged protein appears to be (extremely) overexpressed (and consequently, the morphology of the entire cell is affected).

Does anyone have any tips? I've tried to alter induction conditions of the GFP fusion (a little higher, a little lower) and have tried growing for either 1 hour or 2 hours post induction.

Any advice is most appreciated!
klone
technician-in-training
technician-in-training
 
Posts: 8
Joined: Oct 11 2010 5:45 am

Re: Fluorescence Localization for RFP Fusion but not GFP Fus

Postby CrowSan » Oct 01 2012 10:26 am

Well I am suprised. GFP is normally much stronger than RFP. Are the fusions on the same end of the protein (N or C?)
as it sometimes happens that whether it is a C or N fusion can have an effect on fluoresence (sometimes the flurophore can be "squashed" up against the fusion protein). Also can you see GFP (not a fusions, just GFP) ok on your microscope?
Some GFP variants may have a slightly different Ex/Em profile. Double check the fluorescent microscope you are using to make sure you have the correct filter sets in etc (although I doubt this is the case).
I assume the GFP/RFP constructs are in the same vector? (and nothing silly is happening like the GFP is being run of a eukaryotic, rather than a prokaryotic promoter)....
CrowSan
PI of Posters
PI of Posters
 
Posts: 515
Joined: May 17 2010 7:13 am

Re: Fluorescence Localization for RFP Fusion but not GFP Fus

Postby CrowSan » Oct 02 2012 5:19 am

One other thing... if you are inducing the proteins you may not see fluoresence in the cells until (at least 2 - 3 hours) following induciton. In Eukaryotes 24 hours after transfection is often the brightest...
Also don't forget to image non-transfected cells at both wavelengths (just to make sure the "RFP" signal isn't just auto-fluoresence).
CrowSan
PI of Posters
PI of Posters
 
Posts: 515
Joined: May 17 2010 7:13 am

Re: Fluorescence Localization for RFP Fusion but not GFP Fus

Postby klone » Oct 05 2012 7:41 pm

Hello CrowSan and thanks for taking the time to reply :)

I'll break your reply down into quotes to answer your questions (it's a little easier that way).

CrowSan wrote:Are the fusions on the same end of the protein (N or C?)
as it sometimes happens that whether it is a C or N fusion can have an effect on fluoresence (sometimes the flurophore can be "squashed" up against the fusion protein).


Both fluorphores are appended to the C-terminus of my protein of interest. In both constructs I have engineerd a small (five a.a.) flexible linker between the protein of interest and the flurophore.

CrowSan wrote:Also can you see GFP (not a fusions, just GFP) ok on your microscope?
Some GFP variants may have a slightly different Ex/Em profile. Double check the fluorescent microscope you are using to make sure you have the correct filter sets in etc (although I doubt this is the case).


I have a control vector which can be used to express GFP only in my species of interest. These cells produce good signal when induced.

CrowSan wrote:I assume the GFP/RFP constructs are in the same vector? (and nothing silly is happening like the GFP is being run of a eukaryotic, rather than a prokaryotic promoter)....


No, the vectors are different, and both utilise different inducible prokaryotic promoters. However, we have used the GFP based vector (and also the RFP based vector) to localize a handful of other proteins we are interested in.
klone
technician-in-training
technician-in-training
 
Posts: 8
Joined: Oct 11 2010 5:45 am

Re: Fluorescence Localization for RFP Fusion but not GFP Fus

Postby klone » Oct 05 2012 7:47 pm

CrowSan wrote:One other thing... if you are inducing the proteins you may not see fluoresence in the cells until (at least 2 - 3 hours) following induciton. In Eukaryotes 24 hours after transfection is often the brightest...
Also don't forget to image non-transfected cells at both wavelengths (just to make sure the "RFP" signal isn't just auto-fluoresence).


We have used both vectors to localize a few other proteins of interest (see post above). Generally we would only have to induce for an hour prior to imaging, and any longer than that we see protein overproduction occurring.

This week I made a little more progress by conducting my experiments at 30 deg C. I've heard that GFP matures a little better at lower temperatures, so I gave that a go. I have more cells exhibiting fusion proteins with the desired localization, but we are some ways off the quality of the data set that we gained using using RFP.

Thanks again CrowSan. Any more tips would be appreciated :)
klone
technician-in-training
technician-in-training
 
Posts: 8
Joined: Oct 11 2010 5:45 am


Return to Microscopy and Imaging Techniques

Who is online

Users browsing this forum: No registered users and 1 guest