For specific tips for immunoprecipitation samples:
Load at least 20 ug of INPUT, based on the BCA concentration
We assume that the amount of protein in UNBOUND is the same as INPUT, because very little is being pulled down. So load the same volume for UNBOUND as you did for INPUT, this will ensure you aim at 20 ug of protein in the UNBOUND samples as well
Load the same volume for the IP concentrated sample. You want to make sure the amounts loaded are all relative to each other for good comparison
Load the same volume for the WASHES. You can just load WASH 1, 3 and 5.
3) Antibody IP, concentrated
During IP and sample preparation for western blotting, the IP antibodies that you use have a potential to degrade into their heavy and light chain components. This means, when you load your IP samples into the gel, you are loading not only the protein of interest but also the IP antibody in its whole form or degraded components. The heavy chain is 50 kD and the light chain is 25 kD.
When you detect the primary WB antibody with the secondary, the secondary can potentially bind to the primary but also any heavy or light chains of the antibody used in IP. This means you might see extra bands at 50 or 25 kD, which can be a problem if your protein of interest is that size.
To avoid this problem, we can do one of two things:
1) Use TidyBlot HRP detection reagent - this secondary specifically does not bind to heavy or light chains. Use at concentration of 1:400 in 5% milk in PBS-T.
2) Use a different antibody to the one used for IP, with a different host species. For example if the IP antibody was grown in rabbit, the WB antibody should be raised in mouse or another animal. This ensures that the HRP secondary, which will be mouse or another animal (not rabbit) only binds to the WB antibody in the blot.