Preparing Primary Antibodies for Immunoblots

Preparing Primary Antibodies for Immunoblots

Summary

Make sure the proteins you are identifying are not the same molecular weight. Antibodies are important because They allow for the selective detection of the protein of interest. Typically, a primary antibody is used to specifically bind the protein of interest and a labeled secondary antibody is used for detection so you can see the protein that the primary antibody binds to.

Materials

• TBS+ tween
• Sheet protectors
• Clean trays
• Ice

Method

1. Depending on size of membrane amount of antibody used will change, below is an example from the NEDD8, alpha-actin1, beta- actin, and cullin 7 antibodies
2. Dilute your antibody into a 1.7 ml tube. One tube per antibody. Dilute with TBST + BSA (prepared fresh) dilution factor chart can be found on bench or online

(To determine antibody dilutiontake the amount TBST + BSA (put in ul) and divide by the dilution factor (DF) >>> so (1500ul TBST+BSA / 3000 ul DF = 0.5 ul antibody)

• Beta actin dilution = 1.5ml of TBST + BSA and 0.5 ul of antibody [DF is 1:3000]
• Alpha actin dilution = 1.5ml of TBST + BSA and 0.5 ul of antibody [DF is 1:3000]
• Cullin 7 dilution = 1 ml of TBST + BSA and 1 ul of antibody [DF is 1:1000]
• Nedd8 dilution = 1 ml of TBST + BSA and 1 ul of antibody [DF is 1:1000]
• NOTE: Primary antibodies are expensive so use/ make only what you need

1. Prepare new sheet protector bags for the antibodies, one per membrane
2. After the hour on the rocker, take the membrane quickly and carefully into the sheet protector and seal leaving one side open for the antibody (leave the smaller side open)

Cut any excess and place the bag(s) on ice (keep them on ice as much as possible)

1. Vortex the antibody tube and add the solution to the bags via pipette. Place bags on ice until you are done filling
2. Pull any bubbles on the membrane out by carefully pinching the membrane in the bag
3. Seal the last side and cut any excess
4. Place new antibody bag into a blue container/ tray found on the bench
5. Tape down the bag and place two rollers on top. Make the surface as flat as possible
6. Place tray into the cold room (-4) on the rocker overnight. Ensure the rollers are rolling, add more tape if needed