Western Blot Protocol: Immunodetection - Indirect, Direct

Immunodetection – Indirect

  1. Place membrane into blocking solution for at least 2 hours at rT or overnight at 4°C . Use a sufficient volume to keep the blot fully covered, with gentle agitation throughout .
  2. Rinse the membrane briefly in washing buffer.
  3. Incubate with primary antibody diluted in wash buffer or blocking buffer (an antibody concentration of 1-10 µg/ml is generally acceptable) . Incubate overnight at 4°C, or for 2 hours at room temperature . Use a sufficient volume to keep the blot fully covered, with gentle agitation throughout .
  4. Rinse the membrane in wash buffer (3x10 minutes), with gentle agitation and a sufficient volume to keep membrane well covered.
  5. Add appropriate enzyme conjugated secondary antibody diluted in wash buffer or blocking agent, and incubate for 1 hour at rT with gentle agitation in a sufficient volume to ensure coverage . (*No azide in the buffers if the secondary is labeled with HRP)
  6. Incubate the membrane with gentle agitation as follows: 4x5 minutes in wash buffer, followed by 2x5 minutes in PBS or TBS.
  7. Drain excess PBS/TBS from the membrane and transfer to appropriate enzyme substrate solution and incubate for time period recommended by manufacturer to visualize protein bands.

 

Immunodetection – Direct

  1. Place membrane into blocking solution for at least 2 hours at RT, or overnight at 4°C . Use a sufficient volume to keep the blot fully covered, with gentle agitation throughout.
  2. Rinse the membrane briefly in washing buffer and then incubate with labeled primary antibody diluted in wash buffer or blocking buffer (a concentration of 1-10 µg/ml is generally acceptable) . Incubate overnight at 4°C, or for 2 hours at room temperature. Use a sufficient volume to keep the blot fully covered, with gentle agitation throughout.
  3. Wash the membrane in a sufficient volume and with gentle agitation as follows: 4x5 minutes in wash buffer and 2x5 minutes in PBS or TBS. 
  4. Drain excess PBS/TBS from the membrane and transfer to appropriate enzyme substrate solution and incubate for time period recommended by manufacturer to visualize protein bands. 

 

 


Protocol: Gel Electrophoresis & Protein Transfer by Electroblotting Chapter 6: Western Blot Troubleshooting