Hi, Ying! The appearance of two H3K4me3 bands specifically in the mutant sample could be due to several underlying reasons. One possibility is that the mutation alters post-translational modifications on the protein (e.g., phosphorylation), which can change the protein’s migration on a gel and result in a distinct band. Alternatively, the mutation may affect splicing, leading to the expression of two isoforms of the H3K4me3-marked protein, each migrating differently during electrophoresis. Finally, the mutation may influence chromatin structure or the interaction with binding partners, affecting how H3K4me3 is distributed/ stabilized and resulting in multiple detachable forms. To further investigate, it could be useful to perform mass spectrometry on both bands, use total H3 antibodies for comparison, or test with antibodies specific to other methylation states like H3K4me1 or me2. (Written by AI for ease, reviewed and approved by me).

It seems like the appearance of two bands in your mutant sample could be due to post-translational modifications or alternative forms of histone H3 in the mutant strain. The larger band might represent a modified version of H3K4me3, possibly due to a modification such as acetylation or phosphorylation, which could affect the migration pattern on the gel. Another possibility is the presence of a truncated or alternatively spliced variant of histone H3 in the mutant. Since you confirmed the consistency of total H3 levels with the control experiment, this suggests that the issue is specific to H3K4me3 in the mutant. You may want to consider investigating other post-translational modifications or performing a mass spectrometry analysis to confirm the nature of the additional band. Hope this helps.

I have a follow-up question regarding the use of histone as a loading control. If I observe two bands for histone on my blot, should I consider both bands for normalizing my protein sample? Would this approach be accurate and appropriate? Ping Wu Nichole McTurk Cubbage

If you detect two bands for histone, it's important to first determine whether both bands represent genuine histone isoforms or post-translationally modified forms (e.g., acetylated or methylated variants), rather than degradation products or non-specific binding. If both bands are biologically relevant and represent consistent isoforms across all your samples, then summing their intensities for normalization may be acceptable, provided that the ratio between them does not vary significantly between samples. However, if the relative intensity of the two bands changes across conditions, or if one is likely a modification or degradation product influenced by your treatment, including both could introduce bias. For the most accurate normalization, it is ideal to use a single, well-characterized histone band that shows stable expression across all experimental groups. If uncertainty remains, validating with an additional loading control (like total H3 or an unrelated housekeeping protein) may help confirm consistency. **Disclaimer- I used AI to help answer this (:

Thanks for all your answer. A year later, i repeated this experiment and found that only one band appeared. I only changed the antibody, but it still tje same product with the same catalog. Such a mystery.