Nuclear staining instead of cytoskeleton - (Aug/12/2006 )

Hi there,

I just did some immunochemistry with an antibody against a protein that is known for binding to the cytoskeleton and does not have a NLS. However, my results show big lumps in the nucleus giving a very strong signal and a much weaker signal from the cytoskeleton.

Does anyone have any idea of what might be wrong here?

Thanks,

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What is NLS?

Did u have a positive control for the antibody? How does it look like ?

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how did you fix the cells?

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Hi scolix,

NLS stands for "Nuclear Localization sequence". I do not expect to find this protein in the nucleus if it does not carry a NLS.
What would you call a positive control? The cells I did an immuno staining with are supposed to contain a lot of this protein already.

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Hi gene-hunter-1,

I fixed them with PFA 4% and permeabilized them with triton 0.1%.

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I dont know if a western blot using cytosol and nuclei fractions will help
probe what may go wrong. Any chance that this is not the antibody you expected to be?

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With positive control, I meant if u had tried staining cells before which gives the right pattern of staining.
But since u wrote that these cells contain lot of this protien, i would assume it to b.

I would agree with genehunter on trying out western but subcellular fractionation is not very clean but u could get some idea abt the presence of the protein in the nucleus or cytosolic fractions.

Verify the antibody and the pattern of staining from some papers.

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How does your DIC look?? Do the cells maintain theri integrity???

I think you might want to try your fixing with PF and cold methanol. Sometimes trition can polymerize actin/tubulin filaments. So what you might be actually seeing as lumps in ur nucleus might be clumps of cytosolic or cell membrane fragments.

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I might try a western blot...
I got the antibody from another lab but I trust it to be ok though its concentration is very low (double purification) and I dilute it by 1/20.

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Hi,
I will contact the lab that sent me this antibody to find out more about the pattern of staining

Thanks

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