GFP microskopy and localization - (May/02/2014 )

Hello!

I need some help on how  to use GFP to tag ABC transporter ABCD4, and then use this comparing with some other colour of flourescence (let say red) which will tag ABCD1 transporters which are known to be in peroxisomes. Then I need to compare this two to find out if ABCD4 are also in peroxisomes.

I don't know how to start and what to do.

I was thinking that i would insert sequence of GFP into the sequence of ABCD4 which is also known. That's how i would see the GFP where exactly in the cell it is also at the same time i would do the same only with red fluorescent protein to localise ABCD1 then i would use a computer to compare the two photos at the same time.

I will try this on cell cultures of human brain, liver and adrenal glands.

Is this the right thinking did i miss anything?

Can anyone help me?

Thank you!

Maja

Maja Jurjevec on Fri May 2 14:56:34 2014 said:

Hello!

 

I need some help on how  to use GFP to tag ABC transporter ABCD4, and then use this comparing with some other colour of flourescence (let say red) which will tag ABCD1 transporters which are known to be in peroxisomes. Then I need to compare this two to find out if ABCD4 are also in peroxisomes.

 

I don't know how to start and what to do.

I was thinking that i would insert sequence of GFP into the sequence of ABCD4 which is also known. That's how i would see the GFP where exactly in the cell it is also at the same time i would do the same only with red fluorescent protein to localise ABCD1 then i would use a computer to compare the two photos at the same time.

 

I will try this on cell cultures of human brain, liver and adrenal glands.

 

Is this the right thinking did i miss anything?

 

Can anyone help me?

 

Thank you!

Maja

Yes,

thats how you can do it.

You have to , as you mentioned, add GFP to protein 1 and for example RFP to protein 2.

BUT what is important here: both fluorophores have to be compatible... So you have to check this first!

Check what kind of equipment you have, what kind of fluorophores you can use and how they are "connected" to each other (overlapping spectrum, emission,..) 

You have to make sure you can use both fluorophores with your microscope and that you can detect them well enough.

And you add them to the sequence using a linker. Its not always easy to determine what the best way is: N terminally tagged or C terminally tagged, codon optimized or not, monomers or not.... 

Keep also in mind that GFP itself also (passively) gets translocated to the nucleus itself.

BTW: its not just about looking at a picture and "looking" at a "color" and comparing them like that...

It involves a lot of statistics and microscopy experience... I hope you do have people around you that know more about this if not its pretty much impossible to do this.

Thank you!

I just have to do this hypotheticaly for my school project so i won't actually work on it.

Thank you very much!

Maja