Use membrane extraction kits or try to modify your buffer since RIPA buffer is not ideal for recovery of membrane proteins, especially if they are heavily glycosylated like E-Cadherin.

For example Add 1% Triton X-100 or NP-40 for milder lysis (preserves interactions better). Include 0.5–1% SDS (stronger solubilizer for transmembrane proteins) — but be cautious as it can interfere with quantification. Include protease and phosphatase inhibitors.

Also consider changing lysis time, depending on tissue size do trial and error method, cold lysis can be done. All the best.

To probe for E-Cadherin in a Western Blot:

1. Prepare your protein lysate and run SDS-PAGE.

2. Transfer proteins to a PVDF or nitrocellulose membrane.

3. Block the membrane with 5% non-fat milk or BSA in TBST to prevent nonspecific binding.

4. Incubate the membrane with a primary antibody specific for E-Cadherin (usually overnight at 4°C).

5. Wash the membrane with TBST.

6. Incubate with a suitable HRP-conjugated secondary antibody (1-2 hours at room temperature).

7. Wash again and then detect the protein using chemiluminescence substrate.

Make sure to optimize antibody concentrations and incubation times for best results.

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