TECHNOLOGY

1. Why Use Protease Inhibitors When Preparing Tissue Homogenates or Cell Lysates for ELISA?​​

When preparing tissue homogenates or cell lysates, endogenous proteases (e.g., serine proteases, cysteine proteases, metalloproteases) are released, which can cause:

① Target Protein Degradation: Degradation of the target protein leads to ​false-negative results.

② Protein Fragment Interference: Short peptides generated from degradation may non-specifically bind to ELISA antibodies, causing elevated background OD values.

Consequences of not using protease inhibitors:

① False-Negative Results: Reduced target protein quantity impairs detection accuracy.

② Poor Data Reproducibility: Variability in protein degradation across samples leads to inconsistent results.

③ Elevated Background OD: Non-specific binding of degradation products interferes with antibody recognition.

④ Sample Instability: Residual proteases degrade proteins even at -80°C, particularly in tissue homogenates.

Therefore, during the sample preparation process, the use of protease inhibitors can effectively protect the target protein and ensure the reliability of the experimental results;

2. Can RIPA Lysis Buffer Be Used for ELISA Sample Preparation?​​

Not recommended. RIPA lysis buffer contains surfactants like SDS, which disrupt antigen-antibody binding and compromise ELISA accuracy. Using buffer systems specifically validated for ELISA to ensure stability and reliability.

3. Recommended Protease Inhibitors and its Concentrations​

PMSF (Phenylmethylsulfonyl Fluoride) is the recommended inhibitor.

PMSF is usually stored at a concentration of 10 mg/mL, and its effective concentration is 17-174 μg/mL.

Tissue homogenization preparation: 1 g tissue : 9 mL 0.01M PBS (pH=7.4) buffer. It is recommended to add 17.03-177.08 μL of PMSF. The specific amount can be adjusted according to the actual operation concentration, and it is sufficient to keep it within the effective concentration range.

Cell extracts: If 150-200 μL of 0.01M PBS (pH=7.4) buffer is used to resuspend the cells, it is recommended to add 3-35 μL of PMSF. The specific amount can be adjusted according to the actual operation concentration, and it is sufficient to keep it within the effective concentration range.