invivoVibrio

Samples are prepared as I describe in my first post.  I add 1:1 2x laemmli buffer (4% SDS, 20% glycerol, .004% bromphenol, 125mM Tris-HCl, pH 6.8, BME added to 10% before running), boil for 5 minutes (heating to 70 degrees for 10 minutes does not change the result), and run.

Blocking is with 5% milk in PBST, with .05% Tween.  Washes are done in PBST, secondary is goat anti-mouse from SCBT.  I can add more Tween to the buffers, and remove it from the blocking, but I'm not sure it would help.  Since I'm not seeing a strong actin band, I think something's going wrong before the staining.

Alright, the reducing agent was part of the problem, but not the whole thing.  My bands are a lot sharper and more consistent now, but that's about it.

I've attached an image to show what I'm looking at.

The blots are arranged as mirrors around the ladder in the middle (I was running low on ladder and had to get creative).  I'm mostly interested in the two lanes closest to the ladder, where you see the big fat bands.  I assume the other samples had low protein extraction efficiency, which I'll work out separately.

3D18 and 4N2 are supposed to bind to a 30kDa protein (you can see it on the 4N2 blot, one lane appears to have a PTM of ~8kDa (possibly ubiquitin)).  The 4O18 antibody binds to a 105kDa protein.  Yet all proteins, including actin, show big fat bands where they're not supposed to be.  In this blot, actin barely shows up at all.

Edit: those two lanes are from mouse thymus, fyi.

It could be that the reducing agent is old.  Lots of our reagents are old (though I've bought fresh stocks of almost every reagent used in blotting, just not the BME).  Problem is, our lab has tons of (old) bottles of BME, so I don't know that my lab manager will allow me to buy new stuff.  I'll try it without a reducer (should be fine for actin and other smaller proteins).

I'm pretty sure it isn't native Igs (or any effect of the 2y), since I see the same effect in human cells and rat uterus, and with some primaries, the 60kDa band doesn't show at all (I haven't gotten any bands with Bcl-xl blots yet).