From Western blot to LC-MS on the same membrane

What happens after detection matters too. This workflow follows VerdiBlot from semi-dry transfer and chemiluminescent detection through total-protein staining, band excision and downstream LC-MS identification.

A Western blot can be more than the final image.

This application note follows VerdiBlot through a workflow that begins with semi-dry transfer and chemiluminescent detection, then continues to total-protein staining, band excision, in-membrane digestion and LC-MS identification.

Mouse liver lysate was separated on a stain-free gradient gel using loads from 5 to 100 µg. The gel was divided for a side-by-side membrane comparison under the same transfer run.

The study evaluated:

• compatibility with stain-free gel visualization
• semi-dry transfer
• chemiluminescent detection of GAPDH and histone H1
• colloidal-gold total-protein staining
• manual band excision and tryptic digestion
• downstream LC-MS protein identification

Under the tested conditions, VerdiBlot produced clear GAPDH and histone H1 bands with very low background. After immunodetection, a selected band was excised from the stained membrane and processed for LC-MS, resulting in dozens of confidently identified proteins.

For laboratories connecting Western blotting with downstream protein identification, the note provides the complete workflow, settings and experimental results in one place.

Read the complete application note:
https://www.verdiblot.com/_files/ugd/de6bd3_20fb039744384ea9bc2b5d8a0fdc386f.pdf