5 replies to this topic

[snowchild]

Does anyone tried to watch GFP under microscope after you lysis cells like HeLa?
Or can you see His-tagged GFP under microscope after you purify it using Ni column?

If you can, you don't need to run a western blot (just want to know if expressed or not), right?...
I saw some GFP qutititation kit. So maybe western blot is not necessary.

[bob1]

Potentially, if the concentration is high enough you should be able to see the GFP, but it would probably be very difficult to see as it is likely to be too dilute.  You would be better off putting the lysate in a fluorometer/plate reader with appropriate filters for excitation and emission.

[almost a doctor]

If all you want to know is if the protein is being expressed, could you not just look at the cells under the microscope before lysis?

[snowchild]

Thank you for your response!

Yes, I can look cells under microscope before lysis. Since I want to detect protein after purification, I need to lysis cell.

I will save the transferred membrane after western blot. And I will try to look at it under microscope.

I will also try to read the cell lysate using fluorometer.

Both of the two ways to look at GFP is much faster than western blot, and safer. We use radioactive materia to do WB for quantitation.

I will keep you updated.

[almost a doctor]

Do you have access to a gel-doc system?  You could look at fluorescence in the SDS-PAGE gel before transfer to membrane.  Ideally you want the appropriate filters, however if is just for a quick check: "is it there?", UV light and no filters should do the trick.   Easier than looking at the membrane in the microscope I think, and you can take a picture

[snowchild]

update...
I cannot see GFP band either on gel or membrane using gel-doc.
But I can see TurboGFP band on SDS-PAGE using biorad chemi-gel system.