# Could proteins be transferring through the membrane during the iBlot run

**URL:** <https://scientistsolutions.discourse.group/t/could-proteins-be-transferring-through-the-membrane-during-the-iblot-run/1219>\
**Category:** Western Blotting\
**Created:** [December 9, 2025, 2:14pm UTC](https://scientistsolutions.discourse.group/t/could-proteins-be-transferring-through-the-membrane-during-the-iblot-run/1219 "2025-12-09T14:14:06Z")\
**Posts on this page:** 1\
**Page:** 1

<div class="post-metadata">

**Author:** ![Sarahcanon2025](https://avatars.discourse-cdn.com/v4/letter/s/f04885/32.png) [@Sarahcanon2025](https://scientistsolutions.discourse.group/u/Sarahcanon2025)\
**Post date:** [December 9, 2025, 2:14pm UTC](https://scientistsolutions.discourse.group/t/could-proteins-be-transferring-through-the-membrane-during-the-iblot-run/1219/1 "2025-12-09T14:14:06Z")

</div>

I’m having trouble with my Western blots and wanted to see if anyone has experienced something similar.

I’m running 4–12% or 12–20% Bis-Tris gels (NuPAGE / Bolt), loading ~30–50 µg of protein from cultured cells. For transfer, I’m using the **Invitrogen iBlot 3 Dry Blotting System** , typically running the default 7–8-minute “Mixed MW Proteins” program with PVDF membranes. Blocking, antibody incubations, and washes all follow standard protocols (5% milk or BSA in TBST, ~1 hour block, overnight primary at 4°C, etc.).

The problem: **when I image the blot—even the molecular weight ladder shows absolutely no signal.** I’m using a modern dual-color prestained ladder (e.g., SeeBlue Plus2) to visually monitor transfer. The ladder transfers cleanly to the membrane, but when I develop using **ECL reagents on a digital imager** , there are _no chemiluminescent bands at all_—no ladder, no endogenous controls, nothing.

Could proteins be transferring _through_ the membrane during the iBlot run, leaving me with an empty membrane?