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My beta actin bands are changing


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#1 sialic acid

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Posted 23 April 2014 - 05:22 PM

Hello,

 

Wondering if anyone has observed this phenomena before.  I'm testing a new molecule I made on some cells and when blotting for some proteins I'm interested in, the beta actin control looks like it disappears for the higher concentrations of the molecule.  Now I thought it could have been an error on my part from loading, but the thing that absolutely perplexes me is that the same phenomena is happening across 4 different gels and two different cell lines.  What are the odds that I made the same loading error for only the same drug concentration 4 times?  

 

Has anyone heard of beta actin changing?  I think I did all of the cell lysate/protein normalization calculations correctly.  Of course I'll repeat the experiment again, but can beta actin really be affected that much?  I'm seeing it almost disappear, only at the higher molecule concentrations. I'll try GAPDH, but what if the cell is so stressed that GAPDH will also be affected?  Is there a stain I should do?



#2 jerryshelly1

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Posted 24 April 2014 - 05:30 AM

What does the drug do? Is it a cytoskeletal drug that targets structural proteins?



#3 sialic acid

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Posted 24 April 2014 - 06:30 AM

As far as I know, it shouldn't affect the cytoskeleton.  If it is, it is completely unexpected.  I will try GAPDH as well, but the cells definitely die at higher concs.  If the molecule is indeed inhibiting the cytoskeleton is anyone familiar with what beta actin bands look like when you dose cells with known actin inhibitors?  Are you able to see the bands decrease?



#4 jerryshelly1

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Posted 24 April 2014 - 06:41 AM

Its a weird result for sure. Actin is extremely stable and thus makes an excellent loading control for western blot. Maybe your drug is activating something that degrades actin? What pathway is your novel molecule supposed to be involved in?

 

I forgot to ask you, are you seeing a signal from your target protein? or is the the blot completely blank?



#5 mdfenko

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Posted 25 April 2014 - 04:01 AM

do you see a high molecular weight band form? or something that stays at or near the top of the gel?

 

maybe the actin is polymerizing (as it would when salt is added) in an sds resistant way.


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#6 sialic acid

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Posted 25 April 2014 - 11:39 AM

Hmm well, ran GAPDH and they looked perfect.  Definitely doesn't seem to be a loading issue.  Didn't notice any bands at the top, and for now I really don't want to reveal how the molecule works or what pathway it is targeting since it is quite novel.  



#7 jerryshelly1

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Posted 28 April 2014 - 05:32 AM

just curious... What does your cell look like when you reach the drug concentration that results in a loss of your beta actin bands?






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