8 replies to this topic

[Darlidada]

Hello, 

 

I've a problem , i did an overexpression with fusion protein containning a transcription factor with gfp-G4

I did my transfection on HEK cells with lipofectamine. Once with GFP alone ( from another compagny) and the one from (Biorbyt) I checked after 24 hours of transcription and i saw in each plate the green signal that means the transfection works.

But then i did my western blot . I used the antibody GFP roche, cat. no. 11 814 460 001 and i didn't find any signal for it on my extract. Then my boss said your transfection does't work, but 2 of us saw the green signal by microscope.

I'm thinking maybe this antibody can't reconize  the GFP, when it's associated (fusion) with another factor

Do you have any idea m to solve this problem.

 

THANKS

[CPRES]

I hope you had a GFP negative transfection control to make sure green signal is really true. Also that you had good positive control in your western blot (using known GFP+ cells in similar number) to make sure your western works. Once you can confirm these two, see if your fusion protein is not truncated for the epitope recognized by roche antibody. Also when you say you saw green signal, do you mean focal or everywhere? If focal, western is not going to detect it. In that case, I suggest flow sorting of GFP+ cells before doing western. Your idea (maybe this antibody can't reconize  the GFP, when it's associated (fusion) with another factor) is valid, but only after you exclude other possibilities.

[Darlidada]

I hope you had a GFP negative transfection control to make sure green signal is really true. Also that you had good positive control in your western blot (using known GFP+ cells in similar number) to make sure your western works. Once you can confirm these two, see if your fusion protein is not truncated for the epitope recognized by roche antibody. Also when you say you saw green signal, do you mean focal or everywhere? If focal, western is not going to detect it. In that case, I suggest flow sorting of GFP+ cells before doing western. Your idea (maybe this antibody can't reconize  the GFP, when it's associated (fusion) with another factor) is valid, but only after you exclude other possibilities.

Hello CPRES , 

Thanks for your reply. I don't have a negative control for GFP , what do you mean?  For the positive control, i have it. but the plasmid is from another compagny ( i found it already in my new lab) .

For the green signal, i saw at both plate (gfp alone or gfp fusion) some positive cells , i mean green cells. But i observed that there's more efficiency on GFP alone . But it's still there

[CPRES]

 

I hope you had a GFP negative transfection control to make sure green signal is really true. Also that you had good positive control in your western blot (using known GFP+ cells in similar number) to make sure your western works. Once you can confirm these two, see if your fusion protein is not truncated for the epitope recognized by roche antibody. Also when you say you saw green signal, do you mean focal or everywhere? If focal, western is not going to detect it. In that case, I suggest flow sorting of GFP+ cells before doing western. Your idea (maybe this antibody can't reconize  the GFP, when it's associated (fusion) with another factor) is valid, but only after you exclude other possibilities.

Hello CPRES , 

Thanks for your reply. I don't have a negative control for GFP , what do you mean?  For the positive control, i have it. but the plasmid is from another compagny ( i found it already in my new lab) .

For the green signal, i saw at both plate (gfp alone or gfp fusion) some positive cells , i mean green cells. But i observed that there's more efficiency on GFP alone . But it's still there

 

Negative GFP control means cells transfected with empty vector or non-transfected cells. Just to make sure you don't have auto-fluorescent or previously transfected cells. GFP generally does not change, but using same plasmid transfected cells for western is best. "Some positive cells", is not going to cut it. You need a respectable number of GFP positive cells in your cell lysate to see on western. I think therein lies your problem. Improve transfection efficiency or sort for GFP+ cells.

Edited by CPRES, 12 January 2015 - 08:59 AM.

[Darlidada]

Thank you Cpres. I've already design my new experiment and I planed to have no transfection/gfp alone or fusion gfp.
I'll see.
About the transfection, I'm pretty sure that it works because I've detected the protein fusion via another antibody (against my transcription factor at higher weight that's mean the molecular weight of my protein + Gfp molecular weight) but I can detect GfP . And that's the real problem.So do u think that my hypothesis about the antibody could be true ? and how can I know it ? I checked on their website but this catalog number isn't found

[CPRES]

Thank you Cpres. I've already design my new experiment and I planed to have no transfection/gfp alone or fusion gfp.
I'll see.
About the transfection, I'm pretty sure that it works because I've detected the protein fusion via another antibody (against my transcription factor at higher weight that's mean the molecular weight of my protein + Gfp molecular weight) but I can detect GfP . And that's the real problem.So do u think that my hypothesis about the antibody could be true ? and how can I know it ? I checked on their website but this catalog number isn't found

You got to have a positive control for GFP on the same western blot. If that works and your fusion doesn't (while fusion protein gives band with your protein-specific antibody), your hypothesis may be right. Then it could be due to loss/mutation of the epitope (you can call the company to find out what exact epitope they are using). Conformational masking of the epitope does not happen in denaturing SDS-page gel.

[Darlidada]

Hi 

 

I still have the same problem

i changed my lysis buffer , and now my gels are really clean. i did a new transfection with younger Hek cells

I'm able to detect the fusion protein , by specific antibody  but no detection with the GFP antibody. He detects only on gfp alone condition 

I did a condition : without transfection  and there's no GFP . I changed the antibody, i used a new one from the same compagny, to avoid if juste one aliquot could have a problem, but it's not the case. I called lifetechnology about the antibody and they said there's no reason that it doesn't work especially because it's designed for detecting fusion protein with gfp , on western blot 

I'm thinking that my transfection works but i can't understand why i can't detect it .

 

 

Please share your hypothesis 

[Darlidada]

I solved my problem. My fusion protein was tGFP like Turbo GFP . that's why we didn't detect it . Now everything is doing better . Thanks a lot

[CPRES]

Thanks for the feedback!