Hi Harleen Kaur . The 85 and 50KDa bands are probably the active forms of MMP-2 and MMP-9. The 150KDa band could be homodimers of MMP-9. The higher MW MMPs are usually reduced if you activate the sample with agents like APMA.

Hi Steingrimur,

Thanks for the reply. I'm expecting a protein band of my sample at 150 kDa. My sample is MAb. In addition, I'm not using APMA activation agent in zymogram as the gel was able to detect the metalloproteinases without the addition of the same. It is bit weird as gel can detect low concentrations of metalloporteinases but not MAb which is in higher concentrations.

Regards,

Harleen

A MAb that has MMP-like activty? or an IP of an anti-MMP? In the latter case, any protein-Ab binding is llikely ost when it is in SDS.

Its mmps causing mab fragmentation. Please may I ask whats an ost? Regards, Harleen

Sorry, I typed "lost".

If you are looking at MAb fragmentation of the intact 150kDa, then regular SDS-PAGE will be better than zymography.

Unfortunately, SDS-PAGE won't detect the MMPs at very low concentrations. The advantage of zymogram is it shows clear bands (digested bands) due to the presence of MMPs. And I'm looking for both MAb and MMPs in a single gel.

I would reccommend to do both since either one does not give you the full information. Be aware though that many serine proteinases also show zones of lysis in zymography.

Hi Steingrimur,

Could you please share the literature which mentions about the activity of serine proteases in zymography?

Regards,

Harleen

Hi, Here are references for detecting trypsin-like and plasmin activities using zymography.

https://ajp.amjpathol.org/article/S0002-9440(10)65635-0/fulltext

http://www.jbc.org/content/274/13/8910.full.pdf

You could include PMSF in your samples to decrease those activities

Thanks for sharing the article. I'm looking for MMPs in recombinant monoclonal antibodies.