4.2 Experimental Tips

Experimental Tips
1. Prepare Appropriate Blocking Buffer or Select Commercial Blocking Buffer Based on Different Detection Methods
Detection Method Recommendation
Chemiluminescence Assay Blocking buffer: TBST containing 5% non-fat milk powder
Fluorescence Assay Blocking buffer: TBS containing 5% non-fat milk powder (Tween has autofluorescence, which may cause high background)
2. Key Points for Blocking
If the optimal blocking buffer concentration has not been verified, first select the concentration recommended by the manufacturer.
If antibodies conjugated with biotin or concanavalin are used, non-fat milk cannot be used. This is because milk contains both glycoproteins and biotin, which will reduce the signal intensity.
Natural proteins contain phosphatases that dephosphorylate proteins at specific amino acid residues, so they are not suitable for blocking phosphorylated proteins. In phosphorylation experiments, phosphatase inhibitors should be added to the blocking buffer. Additionally, it is recommended to use TBS as the buffer to dilute the blocking buffer. For the detection of phosphorylated proteins, non-fat milk should not be used as the blocking buffer, as milk contains casein (with phosphorylated epitopes) that can interfere with anti-phosphotyrosine antibodies and lead to increased background signals.