AGP channel looks strange/different in some sources

[shntnu]

CW asked:

About the AGP channel in source 3. It seems that the AGP channel in source 3 is a bit different from replicates in other sources. For example the drug JCP2022_006612 in source 3 (left) and source 1 (right) respectively:

This also happens to other drugs (not as significant as the previous case though): JCP2022_003029, source 3 (left) and source 2 (right)

JCP2022_110997, source 3 (left) and source 6 (right)

Besides, there are some small bright dots that seem to be noise in the first figure. Do you have some insights about why these happen?

[AnneCarpenter]

The small bright dots I believe are a technical artifact (we usually recommend that researchers spin down the staining solution so any aggregates like this are not pipetted into the wells).

To my eye, the AGP staining looks a bit different likely because the balance among the stains used is shifted from one laboratory to another. There are two stains in this channel: phalloidin for actin and WGA for golgi and plasma membrane. Ideally they are balanced so both are visible nicely. I actually don't know which channel is 'ideal' from this perspective.

Take my comments with a grain of salt! others are more expert than me.

[bethac07]

A couple of things I noticed (sorry delayed) -

My major suspicion is that, as Anne pointed out, it seems like the Golgi (WGA) to Actin (Phalloidin) ratio is suuuuper high. This could be a) balance of dyes initially b) where in Z the channel was taken (both those sources were apparently Phenixes, so we can trace that back) and/or c) we've seen intermittently in some collabs that Phalloidin is breaking down in ways that I think were tentatively but not ever fully traced back to the solvent the phalloidin was in (DMSO vs Methanol, IIRC)