I transfected pcDNA and Eprotein in 293T cells, after 48 hours the supernatant was collected and exosomes were isolated, after lysing the exosomes
, an IP was performed on the CD1d, and the final membrane that was revealed was like this picture I put, it was very unclear and uneven, can anyone tell me why this is?
I've repeated this several times and the bands are this uneven, but the variety in the whole cell is very uniform and nice looking. I'm so confused.
It is kind of tricky to troubleshoot this issue based on the limited information provided. You may want to label the lanes, such as "input", "IP", "IgG control", "exosome marker", and "Positive control", so people can understand the readout better.
Not sure if you use the homemade gels or purchased ones, but the rule of thumb is the pH of the gel may dramatically affect the protein electrophoresis. In addition, you may want to make sure the buffer is not too hot during the electrophoresis and transfer.
Good luck.
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