I prepared protein extracts from freeze-dried samples using ddH₂O (60 mg powder in 300 μL water), then confirmed high protein content via BCA Assay—even after 5× dilution, absorbance exceeded the top BSA standard (2 mg/mL).

However, when I loaded these samples onto a 4-20% SDS-PAGE gel and ran it at 200 V for 45 minutes, I observed no visible bands (Lanes 2–7). In contrast, my control group protein extracts (Lanes 8–11) showed clear, well-resolved bands under the same conditions.

All samples were:

• Boiled in Laemmli buffer with SDS and DTT,
• Centrifuged twice (supernatant used),
• Stained overnight with Coomassie Brilliant Blue.

I'm confused because:

• The experimental protein samples (Lanes 2–7) clearly contain abundant protein (confirmed by BCA),
• Yet they consistently show no bands or even smearing on SDS-PAGE,
• While the control and ladders looks perfectly fine.

I’ve considered a few possibilities—protein aggregation, poor migration, staining inefficiency, or interference from polysaccharides or salts—but I’m unsure which is most likely in this case.

Has anyone encountered similar issues when working with plant-based protein extracts?

Thanks in advance for your suggestions!