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B-actin giving 2 bands?

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#1 Stuck with Ligation

Stuck with Ligation


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Posted 20 May 2010 - 11:04 AM

I have a 10 lane gel and I used B-actin as my housekeeping protein. However, on 4 of my 10 lanes I see a 2nd lane right below the expected B-actin bands. All of the cells where lysed the same way and prepared (including boiling time) the exact same. Last, the cells are all from the same line and the same passage.

What are the reasons that B-actin could give a 2nd band? We use a monoclonal antibody. The two bands are clearly distinguished from each other. If it is non-specific binding why would it show up in certain lanes only? The 2nd band does not correlate to any aspect of the experiment (time, + or - control, etc.).

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