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  2. → Viewing Profile: Posts: snowchild

snowchild

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

In Topic: Protein precipitated while kept at 4C over night after elution from a Ni-NTA col

28 March 2013 - 02:56 PM

I read somewhere (probably qiagen handbook of His-tag protein purification) said imidazole will help protein precipitation. So I suggest dialyze right after you elute proteins.

In Topic: Can you see GFP after lysis mammalian cells?

28 March 2013 - 02:41 PM

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.

In Topic: Partial cleavage of HA (Hemagglutinin) tag?

28 March 2013 - 02:36 PM

Which enzyme do you use before MS? If you use Chymotrypsin, your HA tag is highly possible to be cleaved. Check your HA sequence.

In Topic: C-terminal GST fusion problem in mammalian cell

07 February 2013 - 11:23 AM

Thank you.

I decided to make a new construct with other tag, like GFP and His. Forget about C-terminal GST...

In Topic: Can you see GFP after lysis mammalian cells?

07 February 2013 - 11:21 AM

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.

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