After you have blocked the membrane with your choice of blocking agent, you are ready to add your primary antibody to bind your protein of interest. Every antibody will have a different affinity for its particular protein, and protein abundance will vary between cell-types and experiments. As a result, the concentration of primary antibody should be determined empirically for every antibody.

As a general guide, we suggest beginning with a 1:1000 dilution of primary antibody in 5% BSA in TBST for your western blots. We have found that the vast majority of antibodies work very well at this concentration. For loading controls targeted to housekeeping genes (e.g. GAPDH, tubulin, actin) a 1:5000 starting dilution is usually best.

The secondary antibody will always be directed toward IgG of the species in which the primary antibody was raised. So, if the primary antibody is an anti-GAPDH rabbit antibody, you must use an anti-rabbit IgG secondary antibody for detection of the primary.

In almost all cases, the secondary antibody will be conjugated to Horseradish Perdoxidase (HRP). HRP is an enzyme that catalyzes the conversion of an “enhanced chemiluminescent” substrate into byproducts and light. The reaction is similar to the conversion of luminol to 3-aminophthalate and light. However, enhanced chemiluminescent substrates allow for the detection of extremely minute quantities of protein, making the western blot method extremely sensitive.

  1. Dilute the primary antibody 1:1000 in 5% BSA in TBST.
  2. Remove the western blot membrane from the tray with blocking buffer and place into a fresh, clean tray with the primary antibody solution.
  3. Incubate 2 hours to overnight under gentle agitation on a shaker. If incubating overnight, place the membrane at 4°C.
  4. After primary antibody incubation, wash the western blot membrane 3 time for 5 minutes each with TBST. Be sure to use at least 25 ml of TBST for each wash.
  5. Incubate the washed PVDF memrbane with secondary antibody at a 1:2000 dilution in 2.5% BSA in TBST. The secondary antibody can be diluted further if the signal is too strong. However, for most applications, a dilution of at 1:1000-1:2000 will give the best signal to background ratio for your western blots.
  6. Leave the secondary antibody on for 30 minutes to 1 hour.
  7. Wash the membrane 4 times with TBST with gentle agitation on a shaker. Use at least 25 ml for each wash. After the fourth wash, place your western blot membrane into a fresh, clean tray of TBST.



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