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snowchild

Member Since 03 Feb 2013
Offline Last Active Mar 28 2013 02:55 PM
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Topics I've Started

Can you see GFP after lysis mammalian cells?

06 February 2013 - 02:33 PM

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.

C-terminal GST fusion problem in mammalian cell

03 February 2013 - 10:43 PM

Hi, guys. I have some problem with protein expression in mammalian cells. Any help will be appreciated!!

1. I am expressing a protein which has a GST at the C terminal (must at C terminal) in HeLa cells. Do I need to add ATG before GST gene?
2. Is there any tag that is better (at expression level) at C terminal?
3. Some book says if there is a restriction site (especially BamH I) near ATG start codon, the translation is pool in insect cells. Is that ture in mammalian cells?
4. I use pcDNA3.1. The mannual recommend adding Kezak sequence (G/ANNATGG). Mine is AGCATGC (the last one has to be C). Will that affect translation a lot?


I cannot see a band when doing western blot in HeLa cells... My transfection efficiency is ~50%. I can see anti-GFP band. My GST antibody definitely works. Please help me...

Or anyone has some tricks when express protein in mammalian cells? Thanks a lot!

snowchild

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