Blocking prevents antibodies from binding to the membrane nonspecifically minimizing background signals. Two different blocking solutions (5% BSA solution, 5% non-fat milk solution) in TBST or PBST are the most commonly used.
Choosing an appropriate blocking solution is very important as it affects to the results.
TBS or PBS
TBS and PBS both are commonly used buffers for western blot. However, if you are using alkaline phosphatase (AP) conjugated secondary antibody, you should use TBS than PBS since PBS interferes with AP activity. Moreover, you should TBS for detecting phosphorylated proteins. If you use PBS for detecting that, target signal will be significantly reduced but also you might get uneven band signals.
What is TBST or PBST?
TBST or PBST are buffers with Tween 20. Background signal will be reduced by using TBST or TBST. However, too much of Tween 20 can wash away bound antibody and maybe even antigen from the blot. Thus, PBS or TBS with 0.05% Tween 20 is recommended.
BSA or non-fat milk (skim milk)?
In most of cases, when you look up a manufacturer's protocol of antibodies what you are using, they will mention that what blocking buffer you should use. I highly recommend following the manufacturer's protocol.
For example, most antibodies from Cell Signaling are recommended to be used in 5% BSA blocking buffer. You may get good results with 5% non-fat milk but mostly, you get better results with 5% BSA blocking buffer. I used to use 5% non-fat milk for Cell Signaling antibodies but I often get weird bands (e.g. each b-actin bands' thickness was same but intensity was different). However, when I've used 5% BSA blocking buffer, I was able to solve the issues.
Non-fat milk is cheaper than BSA but also it will show you the results with less background noise. However, non-fat milk shouldn't be used to detect phosphorylated proteins as it contains the phosphoprotein casein. Also, it shouldn't be used for avidin-biotin detection system since milk contains biotin.
BSA is more expensive than non-fat milk but it works better with phospho-antibodies. However, it's not compatible with lectin probes since it contains carbohydrates that can increase background noise.
Blocking procedure
Incubate the membrane in blocking solution for 1 h at RT or overnight at 4°C.
Rinse the membrane with TBST or PBST for 5 min at high speed, 3 times. Note. Change TBST or PBST every 5 min.
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