Multiplex Western Blot Normalization | Single-Channel Images

Multiplex Western blotting removes inter-blot variability, but most 1D software was built for single-channel images. Phoretix 1D combines single-channel captures for total-protein normalization. Try it for free on your own images in your own lab now: https://totallab.com/software/free-we... This tutorial shows per-channel background subtraction, per-channel band detection, and paired-channel normalization workflows for LI-COR Odyssey, Azure Sapphire and Bio-Rad ChemiDoc users. What you'll learn: 00:00 – Introduction 00:49 – How to create multiplex images 01:20 – Choosing image channel order 01:39 – Saving multiplex images 02:12 – Moving between channels 02:24 – Aligning images/channels using the nudge feature 02:55 – Automatic lane detection 03:00 – Automatic band detection 03:10 – Per-channel background removal 04:20 – Per-channel band detection 04:56 – Applying global band detection settings 05:15 – Normalizing multi-channel images 05:38 – How to perform total protein normalization 06:48 – How to get a free trial of Phoretix 1D 05:15 – Outro and resource links WHY THIS MATTERS A multiplex Western blot imaged at 700nm (housekeeping) and 800nm (target of interest) has fundamentally different background characteristics on each channel. Applying a single background subtraction across both channels biases the quantification, often by 10-25%. Phoretix 1D's per-channel approach is the only way to get accurate multiplex quantification. IDEAL FOR LI-COR Odyssey, Azure Sapphire, Bio-Rad ChemiDoc multiplex users Labs running paired antibody + total protein normalisation High-throughput protein quantification (drug screening, time courses, dose-response) Publication-quality multiplex Western blot manuscripts KEYWORDS multiplex Western blot, multichannel Western blot, multichannel TIFF analysis, LI-COR Odyssey analysis, Azure Sapphire analysis, ChemiDoc multiplex, per-channel normalization, paired-channel quantification, near-infrared Western blot, fluorescent Western blot quantification, two-color Western blot, Phoretix 1D multiplex #WesternBlot #Densitometry #ImageJ #ProteinAnalysis