A basic protocol for Western blotting using the Life Technlogies iBlot System

Abstract

This is an efficient and basic protocol for Western Blotting using the iBlot® 7-Minute Blotting System and ChemiDoc-IT imaging system to perform Western Blotting.

Reagents

  • SDS-gel , 12%
  • PBS with non-fat milk , 3% w/v
  • PBS
  • Primary and secondary antibodies
  • Triton-X
  • ECL Plus Western Blotting Detection Reagents (GE Healthcare)

Equipment

  • iBlot System (Life Technologies)
  • ChemiDoc-It Imaging System (UVP Inc)

Instructions

  1. Prepare Triton-X in advance as it is highly viscous and takes a while to dissolve. Solution can be kept for 1-2 weeks at +2 to +8°C.
  2. Block membranes with 3% w/v non-fat powder milk PBS (PBS/tween).
  3. Wash with PBS, 2X20 min.
  4. Incubate membranes with primary antibody for 2 hours at room temperature
  5. Wash 6X3 min in PBS.
  6. Incubate membranes with secondary antibody for 2 hours at room temperature
  7. Wash 6x3min with PBS.
  8. Visualise the immunoreactive bands with ECL plus (GE Healthcare) for 1-10 minutes. Detection is carried out on a UVP ChemiDoc-It, Imaging system, (UVP Inc).
  9. Harvest and lyse transfected cells, in lysis buffer.
  10. Run 20-30 µg total protein on in 12% SDS gels (Invitrogen) and transfer to nitrocellulose membranes using iblot system (Invitrogen).

Citation:
Francisco Mansilla. A basic protocol for Western blotting using the Life Technlogies iBlot System. The Molecular Methods database. Tue, 10/16/2012 - 15:14. Acc.nr 6S2et10.