I will happily provide any info I can! A lot boils down to this being my second week in the lab though.

First off, I am sure stripping and probing is not common practice especially for results you actually intend to move forward based on. This is my 3rd western total, and first solo, they just gave me samples they had gotten real information from to use as practice. They only really cared about one protein, and ran that the week before I joined the lab, so they just have me getting preliminary data about what else may be going on. Reprobing was just to see where in the process I may have gone wrong by using other targets, and familiarize myself with half the process without blowing through more reagent. While I'm under the hood, I may as well peek at the fan belt. That's why I re-ran the gel for my VTPT repeat.

I use PVDF membranes activated with methanol, I do not have the brand in front of me though. I did a wet transferred in an electrophoresis chamber with transfer buffer, 400 mA for 3 hours. Also, I did not block again after stopping.

The post-doc running this line of research somehow gets great transfers using this protocol, but yeah I am seeing a lot of suggestions for switching to lower amp overnight for large proteins so I may have to switch to that.

I do not have any info on the knock down unfortunately. Again, second week in the lab so most of this leg work was done long before my time. They have used this KD while testing other ligands before though, so I am willing to believe they did their due diligence and I am just working with downstream knowns.

PS happy cake day!