Hi An-Yu, BMDMs are notorious for giving low/dirty lysates, so a few practical tips:

• Start with enough cells: you’ll need at least 1–2 × 10^6 cells per lane (~20–40 µg protein). If yield is low already, check culture/harvest first.
• Lyse harder: RIPA is often too mild. Go with 1% SDS buffer or just boil cells directly in 2× Laemmli sample buffer (with DTT), then sonicate to shear DNA. That usually gives a nice clear lysate.
• Deal with viscosity: if your lysate is sticky, add Benzonase or DNase + Mg²⁺, and sonicate.
• Check the blot setup: if your loading control is blank, confirm transfer with Ponceau stain and try a positive control lysate (e.g. RAW264.7). Sometimes it’s not the cells, it’s the gel/transfer.
• Load more protein

Also, scrape BMDMs into Laemmli buffer, boil, sonicate, spin, and load. Works much better than RIPA in my hands!

We provide Protein Extraction and Mammalian Active Protein Extraction Reagent protocol on our website, check them out to see if they help!

Lucian Miles thank you for your advice, I will try this!!