It's possible that the cells could have undergone changes in their machinery, which could affect translation, folding, or post-translational modifications of the antibodies. Cell lines can exhibit genetic instability or undergo phenotypic changes over time, especially when cultured for extended periods or subjected to passaging. These changes could potentially impact protein expression and functionality.

Using a relatively low passage number from the initial commercial stock (P.7) should minimize the risk of significant changes, but it doesn't entirely rule out the possibility. Other factors, such as variations in cell culture conditions, media components, or reagents, could also contribute to the observed issues.

To troubleshoot this problem, you may consider these:

1. Verify the integrity of your cell line by comparing it to a freshly obtained sample from the same commercial source or obtaining a new batch altogether.

2. Assess the stability of your cell culture conditions, including media, supplements, and any other factors that could impact cellular behavior.

3. Explore alternative antibody production methods or cell lines to confirm whether the observed changes are specific to the current system.

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Hi,

one possibility is that you diluted them too much during passaging (never more than 1/10th ) then if a mutant appears and if not producing the antibody is an advantage for it then it can takes the lead on the whole culture .... go back to earlier passages you have in liquid nitrogen