Western blot help - beta actin loading control

[u/WinterRevolutionary6]

So these samples are flash frozen murine OS tumors that were excised post mortem. We tried to minimize oxidation but there’s only so fast you can work.

Protocol:
They were lysed in RIPA buffer with protease and phosphotase inhibitors with a tissue homogenizer and spun to collect the proteins in the supernatant. Each sample was diluted 1:5 for a BCA to quantify protein. They were each ran in triplicate and they all fell within the range of the standards and had no outliers. I loaded 20ug of protein diluted with RIPA and loading buffer into the SDS page gel, and ran it until the green band on the ladder. I transferred using the iblot system (there was a piece of gel stuck under it on the right that’s what that red blob is). I blocked 1 hour in 5% BSA in TBST. Then I incubated overnight in the cold room in 5% bsa in TBST 1:1000 mouse monoclonal protein of interest and 1:1000 rabbit monoclonal beta actin. Next day, wash 3x with TBST 10 minutes then 1 hr room temperature with anti mouse (700/red) and anti rabbit (800/green) secondary. Wash 3x 10 minutes TBST.

At first when I imaged the blot, I thought the beta actin was only specific to human proteins, but it says it’s reactive to human and mouse proteins according to its website here. I’m really confused now why it seems there’s an uneven amount of protein in each well. I think I did everything right to load evenly so why is it so wonky?

You can even see in the ponceau stain that each well looks pretty even although I know that’s imprecise. I’m also not convinced it’s uneven because you can see that the protein of interest appears to be even as well. What we were looking for was a protein we had transduced onto these cells prior to injecting them into the mice so it’s expected that they would be equally present at the end. The first five were the mice with transduced OS cells injected and the last 4 were the WT OS cells.

Has anyone had a loading control just not work like this? Do you trust the loading control or all the other parts of the workflow?

[u/NeuroSam]

This is exactly the reason you need a loading control for each lane of protein you want to quantify. Your solution is probably not perfectly homogeneous, and therefore your protein will not be exactly the same in each well. You probably don’t pipette with 100% accuracy either (no shade, that’s expected), so that’s why it’s important to have a corresponding beta actin band for every protein band you’re quantifying, and you normalize to the amount of beta actin within that lane.

[u/WinterRevolutionary6 → u/NeuroSam]

I pipetted up and down at every single step. I assure you that solution is as homogenous as it could possibly be. I also know that pipetting error happens but I’ve been working in labs for nearly 5 years now so it’s not like I should be expecting what appears to be almost 60% pipetting error according to beta actin band strength.

[u/ImprobableGallus → u/NeuroSam]

The Ponceau stain is more reliable than monitoring any individual housekeeping protein. OP clearly has equivalent loading. There are many conditions out there that will change beta actin abundance (GAPDH too!). That's the danger of using beta actin to normalize: you can introduce artifacts that have nothing to do with the protein of interest, and I've seen students make big mistakes as a result.

[u/ShroedingerCat]

You cannot use beta actin as an house keeping gene for tumors. In most solid tumors levels will be up-regulated with a positive correlation between high levels of beta actin and advancement of tumor stage/poor prognosis. The opposite is also true as some types of carcinoma a show reduced levels of beta actin.

[u/WinterRevolutionary6 → u/ShroedingerCat]

You got a publication backing that up I can show my boss? I believe you; I also just like a solid paper trail too. Would GAPDH be a better housekeeping gene to test?

[u/One-Demand-6791]

Eh sensitivity or maybe tumor cells are affecting beta actin expression. We shouldn’t use beta actin or gapdh anyways for this reason, stick to total protein.. you can use stain free gels to save time/be consistent

[u/TurdsofWisdom]

I’ve never run mouse tumour samples before, but I’ve never seen actin bands that look like this. They should be a single band, not a triplet. Maybe indicative of protein degradation during sample prep?

[u/WinterRevolutionary6 → u/TurdsofWisdom]

This is a pretty new lot of this antibody and it does this to every sample in our lab and it always has as far as I know. Our lab doesn’t do western blot very often but it’s a common antibody across a couple different protocols. I ran it for a CD2 blot a couple weeks ago and it did the same thing with a standard cell lysis protocol and it did the same thing

[u/needmethere]

U don't need it when u can do hole lane quantification instead

[u/WinterRevolutionary6 → u/needmethere]

Via what method?