Does anyone know if there is any condition or modification of protein that will prevent the protein band to be shown on SDS-PAGE gel after silver staining? I recently produced a human protein from insect cells using FLAG peptide as tag, however, after HPLC to purify the protein and nanodrop to measure the concentration, I cannot see any band from that protein on SDS-PAGE gel with silver staining. But there is a good peak and high concentration as indicated by HPLC and Nanodrop (0.2mg/ml stock concentration and I loaded 2ul of the protein into the lane). The molecular marker and positive control protein both showed up as expected on the gel. Both my labmates and I are very confused by this result as we tried running gel several times and still see the same result. We also tried denature gel and native gel but couldn't see anything changed. Please let me know why there is such a conflict result between HPLC/nanodrop and silver stained SDS-PAGE gel. Any idea/thought will be greatly appreciated!
The Nanodrop simply measures UV absorption, thus any compound absorbing in the 280 nm region will interfere. Complement with a standard protein assay like Bradford or BCA to check that there really is protein present.
Run size standards with yuor electrophoresis, if their bands stain correctly you know that the elektrophoresis worked.
Fluorescent staining with RuBPS (Dye hard) is as sensitive as silver, but quicker, cheaper, and a lot less finnicky. I have published a detailed protocol in Meth. Mol. Biol.
Hi Nelson,
I think that your protein may undergo self-cleavage, forming two smaller protein bands. You may still have a native precursor protein, but usually denaturation causes it to disappear when attempted to be detected with Coomassie or other imaging methodes. If you have anti-protein antibodies, try detecting the smaller auto-cleavage samples. You can get more detailed information on the cleavage and mechanism on this site:
https://www.jbc.org/article/S0021-9258(20)79630-4/fulltext
Hi,
Did you confirm that your purified protein is the target FLAG-tagged one by employing any immunoaffinity-based detection?
Assuming your protein is accurately extracted and purified, your elution mechanism is also important. Did you elute your protein by lowering the pH and which compositions of binding and washing and elution buffer did you choose?
Acidic pHs are problematic due to incompatibility with Leamnli sample buffers, you need to stay neutral to slightly alkaline pHs...Developed color before sample loading can indicate something.
At this point, it is better to share information about your sample preparation protocol for PAGE and loading concentrations of your control reference and sample along with purification workflows to get a clue.
At last, as authors indicated BCA or Bradford assays are more reliable to get certain concentrations.
Emir
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