Hope you´re all doing well!
I´m currently in a bit of a situation. I have done Western Blot on primary human monocytes . I´ve tested the usability and approx. parameters used beforehand on THP-1 cell Lysate.
In case of the THP-1 cells, every antibody worked and produced a usable signal. In case of the primary human monocyte lysate it didn´t work. The proteins should be expressed in the lysate and Tubulin reacted as well, just the lanes with the other antibodies didn´t react. There are no bands at all.
I´ve used a RIPA Buffer for lysis, the monocytes were isolated via beads, for the transfer I´ve used a nitrocellulose and for imaging an ECL kit.
Does anyone have a good guess what went wrong/ How i could improve? My current only guess is that either the antibodies may have degraded over freeze/thaw cycles (stored according to the manufacturer) or that the proteins in the lysate did degrade/denaturate over time (produced single use quantities and started the blot max. 3 days later, stored at -80°C).
Dear Fabian Roggatz
Check your target primary antibody is reactive with Human (H). For cross check you can try in other cell lines. And be sure about the presence of the biomarker in your cell line.
Kind regards
AB Bayazid
Dear A.B. Bayazid,
The antibodies should all be reactive to human according to the manufacturer. Is there a varying specifity and resulting variation in intensity?
The lysate in different experiments originiated from different donors, I think that would exclude the possibility of diseases (if you meant that by biomarkers).
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