The markers on the far left are 180 kDa, 130 kDa, 100 kDa, and 75 kDa. The three lanes are control group 1 and treatment group 2, respectively. Each group is separated by a marker. The primary antibody used is a TFR antibody, and the secondary antibody is abcam Goat Anti-Rabbit IgG H&L. The Western blotting results now show a lot of spurious bands. I'm not sure what's causing this or how I can improve it.
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The predicted protein band size for this protein is 85 kDa, but dimers of 150-200 kDa may also be observed.
It is recommended to add a positive control, such as HeLa cell lysate, K562 cell lysate, or Raw 264.7 cell lysate.
Additional experimental optimization suggestions are as follows:
1. Use a 0.45 μM membrane transfer buffer.
2. 10% methanol is sufficient for transfer buffer.
3. This image does not clearly separate the large proteins; you may want to run more with an 8% separation gel.
4. Use the same antibody diluent as the blocking buffer; 5% skim milk powder is recommended.
The answer to this question comes from MCE Technical Support.
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