Preparing Secondary Antibodies for Immunoblots
From SDMRC
Preparing Secondary Antibodies for Immunoblots
Summary
As mentioned the secondary antibody is used to visualize what you are looking for, it is an amplificator. We also use the secondary to clean the membrane of excess background. Later when a reagent is added it makes a signal that you can see.
Materials
- TBS+ tween
- Sheet protectors
- Clean trays
- Ice
Method
- Rinse rectangle trays with DI water, and fill with TBS tween
- After the overnight incubation remove the membrane from the bag(s) and place into the tray with solution
- Place on the rocker for 10 minutes minimum. Repeat this wash step two more times. The membrane can stay in TBS tween longer as long as it is in solution
- Prepare new sheet protectors for each membrane
- Depending on what the primary antibody was prepared in determines you secondary antibody. You can figure out what the primary is prepared in from the label or looking up the lot number. The secondary will be opposite or anti of that species.
In this example: Alpha-actin1: primary prepared in mouse so secondary is anti-mouse beta-actin: primary prepared in mouse so secondary is anti-mouse Cullin 7: primary prepared in mouse so secondary is anti-mouse Nedd8: primary prepared in rabbit so secondary is anti-rabbit
- Secondary antibodies can be found in the fridge door. The dilution factors can be found on the bench but for mouse and rabbit the factor is 1:1000.
- Prepare secondary antibody on ice, determine amount needed by the size of the membrane. Same equation as primary: amount of TBST + BSA / DF = amount of antibody
- Take the membrane into the sheet protectors quickly and seal leaving a small side open
- Pipette the amount of antibody solution needed to each bag. Place filled bag on ice when you are filling the other bags
- Get rid of air bubbles and seal
- Place in back on the rocker with rollers *at room temperature* for 1 hour
- After the 1 hour, wash the membranes again three times in TBST for 10 minutes
