Western Blot Protein Expression Detection and Complete Workflow

A one-stop experimental framework covering sample lysis, protein quantification, SDS-PAGE, membrane transfer, antibody incubation, signal acquisition, and data analysis.

Experimental Objective

To detect the expression level, molecular weight, and relative changes of specific proteins in complex samples, and to investigate protein activation status using modification-specific antibodies.

Applicability

  • Samples: Cell lysates, tissue lysates, purified proteins, and adapted bodily fluid samples
  • Species: Human, mouse, rat; other species require antibody reactivity verification
  • Difficulty and Time: Medium, usually about 1–2 days

Standard Workflow

  1. Lyse, clarify, and quantify protein
  2. Load equal amounts for SDS-PAGE
  3. Select PVDF or NC membrane and complete transfer
  4. Block and incubate with primary antibody
  5. Wash and incubate with labeled secondary antibody
  6. Chemiluminescence or fluorescence imaging
  7. Normalize and perform statistical analysis within the linear range

Required Materials

Equipment

  • Gel electrophoresis and blotting system
  • Shaker and imaging system
  • Protein quantification instrument

Reagents

  • Lysis buffer and protease/phosphatase inhibitors
  • Protein quantification reagents, gels, and molecular weight standards
  • Transfer, blocking, and washing buffers
  • Primary antibodies, secondary antibodies, and detection reagents

Consumables

  • PVDF or NC membranes
  • Centrifuge tubes, pipette tips, incubation boxes, and personal protective equipment

Required Controls and QC

  • Positive control, negative or knockout control, loading control, and secondary antibody blank
  • Even membrane transfer, target band near expected molecular weight
  • Signal for quantification must not be saturated

Common Problems

If no band is present, check sample integrity, transfer, antibody reactivity, and exposure settings; if background is high, optimize antibody concentration, blocking, and washing conditions; if bands are smeared, reduce loading volume and confirm complete sample denaturation.

Shop by Material Role

Note: The specific Bill of Materials (BOM) will be supplemented after the actual products are inventoried and antibody reactivity, equipment compatibility, and specifications are reviewed.