Dear esteemed researchers,
I have recently conducted Western blot experiments to detect uPAR (CD87). The whole-cell lysate samples I used were obtained from four human glioblastoma cell lines (U87, U251, LN229, LN319) and two mouse glioblastoma cell lines (GL261, G422). According to previous literature data and the recommended molecular weight by the antibody supplier, the expected molecular weight of the target protein is around 50 kD. However, the band positions in my Western blot are not aligning correctly (considering that my experimental conditions should not have fundamental errors, as I have conducted other protein Western blot experiments where the band positions correspond to the correct molecular weights).
Has anyone else conducted similar experiments? Could you please provide some advice on why such results might occur? I appreciate your valuable time and generous assistance.
Thank you!
Dr. Yang, in the context 6 cell lines were mentioned but 8 lanes were demonstrated in the figure. What the extra two samples are? Are they positive or negative controls?
The predicted molecular weight of the protein is based on its amino acid sequence which could be distinct from its real molecular weight. uPAR can be post-translationally modified and appears at different positions on the membrane.
So, I have two suggestions:
1. Overexpress or knock out the uPAR in one or two cell lines and use them as controls to figure out which band represents the uPAR in your cell lines. I personally prefer overexpressing, because the overexpression can be visualized by tags or protein bands.
2. use two different antibodies to cross-validate the antibody's quality.
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