10 replies to this topic

[Curtis]

we are transfecting BHK cells with GFP vectors using both Lipofectiamine and Calcium Phosphate methods but we cannot see any expression under direct fluo. microscope.

I used to do transfection 5-6 years ago with inverted fluo. microscope and now I'm using the same methods but I cannot see any green GFP in the cells under direct fluo. microscope.

I do ImmunoFluorescence experiments with the same direct microscope and get sharp pictures but I'm not sure if the same IF methods (e.g. growing cells on coverslip, fixation and putting on glass slide) can be applied to EGFP expression studies.

we are planning to lyse our cells and do western blot with anti-EGFP. Is a T25 flask enough for lysis and detection of EGFP expression after 24-48 hours?

can we also do Flowcytometery?

[little mouse]

our EGFP transfected cells are observed under inverted fluorescent microscope (in the growing medium). There is no problem (after 24-48h of transfection).
You can see them by FACS too.
Are you sure you are using the right promoter (naive question)?

[Curtis]

our control is pEGFP.N2...even that one doesn't show green color!

[little mouse]

If you have access to a flow cytometer, it would be faster and more easy than western-blot.
I don't understand why you can't see the cells under your microscope (especially if you put them on glass slide).
if flow cytometer shows no fluorescence, I would conclude that your transfections were bad.

[little mouse]

are you fixing your cells with PFA?

[memo]

Transfect always the intern control CMV-eGFP and check with FACS analysis for percent cells. Within 24h I have enough expression to detect GFP even in primary cells which are harder to transfect. For optimal results I check after 48-72 hours! What is your transfection method exactly?

[Curtis]

little mouse:
I fix with 3% formaldehyde as I always do for my IF experiments
so you recommend Flowcytometery. just as I thought but I don't know much about flowcytometery and FACS.

memo:
we follow invitrogen's protocol for Lipofectamine. just a very routine protocol

I'm going to get a phMGFP vector from a friend of mine tomorrow to re-clone our gene in it. the current pEGFP.N2 that we have is from an old stock of one our post-docs who left the lab already. i'm not sure if it's a good vector. so i'm gonna try with Promega's phMGFP tomorrow.

[little mouse]

I think I get it.
Reduce the formaldehyde concentration to 0.5-1% because it reduces GFP signal and increases background.

[swanny]

Naive question, but is the construct in the correct frame? I only ask because we had two occasions last year where non-expression of proteins came down to a (missed) frameshift.

[scolix]

Curtis, on Mar 17 2009, 09:56 AM, said:

Is a T25 flask enough for lysis and detection of EGFP expression after 24-48 hours?

we use lysate from a single well in 24 wells to detect GFP. Better to wait atleast 48 hrs before checking expression.

[Curtis]

little mouse, on Mar 18 2009, 02:29 AM, said:

I think I get it.
Reduce the formaldehyde concentration to 0.5-1% because it reduces GFP signal and increases background.

thanks, we'll do that next time. we're gonna do many experiments this week and hopefully we'll get the result we want.

swanny, on Mar 18 2009, 03:03 PM, said:

Naive question, but is the construct in the correct frame? I only ask because we had two occasions last year where non-expression of proteins came down to a (missed) frameshift.

pEGFP.N2 is a commercial plasmid from Clontech I think, so it must be in the right frame. I used the same plasmid 5 years ago and had no problem.

scolix, on Mar 21 2009, 10:51 PM, said:

we use lysate from a single well in 24 wells to detect GFP. Better to wait atleast 48 hrs before checking expression.

thank you.