I am working on skeletal muscle synthesis, and I did Western to see p70s6k.
I expected I could see the p70s6k band around 70 kDa, but I saw two unexpected bands below (attached).
I am not sure how to interpret the bands.
Western information that I did is the following:
10% gel__Running 50v for 30min and then 100v for 2h__Transfer membrane (PVDF) for overnight in cold room (36v)__Cell signaling Primary (rabbit)
I would appreciate it if you leave any comment on that.
Sincerely,
Sonny.
Hello Sonny,
The upper band could be the phosphorylated form of p70S6K (as it is a kinase and required phosphorylation for its activation) or it might be derived from its isoform p85S6K (if the epitope is similar).
Hi Mondal, Satyendra Mondal
Thank you for your point of view.
I should figure out what you said.
Best,
Sonny.
Based on the information provided, it is difficult to say with certainty what the two unexpected bands below the expected p70s6k band represent. However, there are several possibilities to consider.
First, it's possible that the two lower bands are isoforms of p70s6k. This could be confirmed by performing further analysis such as mass spectrometry or immunoprecipitation to identify the specific isoforms.
Another possibility is that the lower bands could be non-specific binding or background noise. This could be due to factors such as incomplete blocking of the membrane, inadequate washing, or non-specific binding of the secondary antibody.
Finally, it's possible that the lower bands represent degradation products of p70s6k. If this is the case, it could be helpful to assess the level of degradation by comparing the band intensity over time.
To determine the cause of the unexpected bands, it may be necessary to repeat the experiment with additional controls or modifications to the experimental protocol.
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