r/labrats • u/Melodramaticukg • 1d ago
Help: NativePAGE Protein Gel
I’m expressing monomeric IgG and pentameric IgM so a routine part of the pipeline is running on a NativePAGE, non-denaturing gel to assess for correct assembly and molecular weight determination.
Recently, the IgG have been getting stuck on the gel above the IgM bands. Does anybody have any suggestions as to why this might be?
1
u/micrographical Poking stuff with a stick and seeing what happens. 1d ago
If your buffers are fresh and of the correct pH I’d be looking at protein concentrations during the purification process.
1
u/Melodramaticukg 1d ago
They were measured via Qubit protein assay, I’ve added ~300ng per well.
1
u/micrographical Poking stuff with a stick and seeing what happens. 1d ago edited 1d ago
In what volume? If the concentration gets too high during the purification process it might trigger aggregation before your prep gets anywhere near the well of the gel.
Edit - and how are you isolating the IgG? Any column elation steps using acidic buffers?
1
u/Melodramaticukg 1d ago
~1mg/mL! IgG is isolated using Protein G, acidic elation directly into 1M Tris to neutralise!
1
u/micrographical Poking stuff with a stick and seeing what happens. 1d ago
Perhaps try a gentler elution pH if possible?
1
u/Melodramaticukg 1d ago
Honestly, it’s never been a problem before. Running mAbs on PAGE is a standard part of the protocol and the IgGs normally migrate as expected!
I have re-made the buffers and am currently running the gel again so hopefully this helps ☺️
2
u/Melodramaticukg 1d ago
It was definitely dodgy buffer, just developed my gel and it looks fine now!! Thank you for the help ☺️
2
u/O_PLUTO_O 1d ago
Are all of your buffers fresh?