I am conducting a Western Blot probing for CD133 and GAPDH. I use BioRad Stain-free Gels (any kD) and their turboblot accessory with their nitrocellulose mini membranes. My protocol is as follows: Run my samples (~10ug of protein per lane; 30uL of sample with 9uL of Laemmli Buffer; 27uL per well; heat to 65-60C for 10 minutes) at 150V for 1 hour; imaging the stain-free gel using chemidoc MP unit; transfer using the mixed MW 7 minute protocol (checking the gel afterwards); block using 5% milk for 1 hour at room temperature; incubate in primary Ab (CD133 1:500, Gapdh 1:5000) overnight in 4C; Wash with TBST (1 min, 1 min, 2 min, 3 min); incubate with secondary Ab in TBST (1:7500) for 2 hours at room temperature; Wash with TBST (1 min, 1min, 2 min, 4 min); incubate with ECL reagent (total volume of 2mL) for 5 minutes; imaged with auto exposure, but got an error saying the image was too faint, so tried the step feature with 4 images between 10 seconds and 120 seconds of exposure. Same protocol for all three westerns mentioned below.

In the pictures are the results of my run #1 (not a great blot, but the bands show up); in the second picture is the stain free gel image of the run #2, third image is the membrane stain-free image of run#2 and then the final image of run #2 of the membrane. I noticed that for run#2 the stain free gel shows distinct bands and lanes (as did run#1), but I get nothing for the final membrane image. I then did a third run and my run #3 got the exact same results as run #2.

IMPORTANT NOTE: for run #2 and run #3 my samples had thawed due to our -20C fridge being left open and I have no way of knowing how long they were thawed, so my concern is that my proteins degraded, but I thought that they would still show up on the final image, just at different MWs, unless perhaps they were completely degraded and ran out of the gel, but then why would there by bands showing up on the stain-free gel?

So, I guess my question is why would I get lanes and bands on my gel stain-free image and membrane stain-free image, but blank on my final membrane?

Thanks for any assistance.