A step-by-step guide to properly carrying out Western Blot Assays

Western Blot

Western blot is an essential and powerful analytical method. By utilizing a western blot, researchers can detect specific proteins from among a complex mixture of proteins removed from cells. They can be used to estimate the size of a protein of interest and to calculate the amount of protein expression.

LI-COR Odyssey is a flexible, infrared (IR) laser-based instrument that provides improved data quality and sustained sensitivity. It can detect even faint mobility shifts because of protein modifications. It is using infrared fluorescent (IR fluorescent) signals to identify strong and weak bands within a single image, without signal saturation on a western blot. LI-COR can accurately measure changes in protein levels through a ratiometric approach. It has 700 and 800 nm infrared fluorescent detection channels that enable a two-color target analysis and normalization.

By normalizing signal intensities from two IR detection channels, LI-COR Odyssey western blot delivers protein quantification with comparable sensitivity to chemiluminescence. Li-COR Analysis depends on longer wavelengths from near-infrared imaging for sensing strong and weak bands on a western blot to create a low signal-to-noise ratio along with a broad and linear dynamic range.

LI-COR Odyssey provides both chemiluminescent and IR fluorescent protein detection minus the film usage. This elimination gives flexibility, data archiving, developer and darkroom cost savings, and environmental friendliness. The platform enhances the accuracy of quantification greatly and provides flexibility for the blot processing and storage as the sign from the IR fluorescent dyes is robust.


  • Superior sensitivity
  • Wide linear range
  • Multiplexing
  • Broad applicability


  1. Sample Preparation – By using mechanical and chemical disruption, proteins are extracted from biological material. The total protein is measured, and samples are mixed with a buffer before loading them on a gel for electrophoresis.
  2. Gel Electrophoresis – Protein samples are loaded onto a gel; this is where they get divided by size through electrophoresis. Gel electrophoresis can be done in native or denaturing conditions.
  3. Gel Transfer – Proteins are transported electrophoretically to membrane support, where they are taken for subsequent immunodetection.
  4. Membrane Blocking – The membrane is then detached from the transfer stack, positioned in a container, and incubated with a blocking buffer.
  5. Antibody Incubation – The blot is incubated with a prime antibody given for an epitope present on the target protein. After several washes, the membrane is then incubated with a labeled secondary antibody to provide a way of detection.
  6. Detection – After washing the membrane to take out unbound antibodies, the label on the secondary antibody is spent to imagine the protein of interest.

NorthEast BioLab efficiently uses LI-COR Odyssey to provide precise, quick, and robust Western Blot Analysis, In-Cell Western, and Protein Gel Imaging. Our scientists develop dependable bioanalytical methods and implement your sample analysis assay for several stages of drug development. We use LI-COR western blots to precisely identify cell proliferation, apoptosis, cell signaling, and numerous biomarkers, and quantitate proteins in a filled, linear range along with transfer images and data files to clients for convenience and safekeeping for long-term storage.