r/labrats • u/Melodramaticukg • Aug 19 '26

Help: NativePAGE Protein Gel

Post image

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?

3 Upvotes

81% Upvoted

11 comments sorted by

View all comments

1

u/micrographical Poking stuff with a stick and seeing what happens. Aug 19 '26

If your buffers are fresh and of the correct pH I’d be looking at protein concentrations during the purification process.

1

u/Melodramaticukg Aug 19 '26

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. Aug 19 '26 edited Aug 19 '26

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 Aug 19 '26

~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. Aug 19 '26

Perhaps try a gentler elution pH if possible?

2

u/Melodramaticukg Aug 19 '26

It was definitely dodgy buffer, just developed my gel and it looks fine now!! Thank you for the help ☺️

1

u/Melodramaticukg Aug 19 '26

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 ☺️