Question (58030) | Anti-RASSF1a antibody [3F3] (ab23950)

Go to datasheet (ab23950)

Question

Hi,
I'm trying doing WB using ab23950, but I could not detect signals around 40KDa in HS68 and BJ5 cells.
Could you tell me the condition of western blot for this antibody such as which lysis buffer to use, sonication is necessary,
now much protein per lane, nitrocellulose or PVDF membrane etc.
Also, which cell type have you tested besides HeLa cells? Do you think it can be observed in fibroblast cells such as HS68,
BJ cells, NIH3T3 cells?
Also ab97749 antibody's IF staining in HeLa cells shows nuclear staining where this proteins localization is reported
as microtubule localization. Why?

Answer

Thanks for your enquiry.
We do not have a specific protocol for cell extraction when preparing samples to western blot RASSF1a. We do have a good general WB protocol which you might find useful at https://www.abcam.com/index.html?pageconfig=resource&rid=11375.
Based on information on the datasheet for ab23950, there is western blot data showing detection of RASSF1a in Hela cells, mouse brain, liver and kidney extract.
Based on information provided in the SwissProt database, this protein has multiple forms but is expressed in all tissues, but often not in the corresponding tumor cell lines. It may be useful to check the literature on RASSF1a for cell lines that do contain detectable levels.
Since normal tissues commonly contain detectable levels of RASSF1a, you may be interested in our range of tissue extracts. Simply type in the tissue type into the product search bar at the top of our website, and select 'Lysates' as the product type.
Also, the SwissProt protein entry for RASSF1a notes that this protein can change cocalization, from cytoplsam to nuclear, depending on the cell cycle. Again, it may be useful to consult the literature for further details.
I hope this is helpful. Please contact us again if you have any further questions.

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