Anti-RASSF1a抗体[3F3] (ab23950)

The anti-RASSF1a ab23950 recognises the first exon and should therefore discriminate the exon1 deleted mutant to the wild type protein.

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.

Thank you for sending this paper. I have passed this information on to the lab and my colleague provided the following clarification of our testing:

"During the development of this antibody, our western blot result wasn't good in A431 cells, but we could see it more clearly in immunofluorescence. We concluded that it was due to a very low expression level in A431 cells. It's quite possible that it isn't detected in WB or RT-PCR. We couldn't find any conclusive supporting medical reference for expression or non-expression of RASSF1a in A431 cells."


I hope this helps, please let me know if you need any additional information or assistance.

Thank you for your enquiry. This antibody was raised to recombinant full-length hRASSF1a, and not a peptide sequence. The most detailed information we have regarding the epitope of ab23950 is that it specifically recognises the C1 domain (1-340aa) of RASSF1a. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Thank you for providing the customer's detailed protocol, it enabled me to investigate the problem rapidly and thoroughly. There are two main points I would like to concentrate on: 1) levels of the protein may be low in the customer's samples. Following some publication readings I found several references for low levels of RASSF1a in some gastric carcinomas. For example, Byun DS, Lee MG, Chae KS, Ryu BG, Chi SG.Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma.Cancer Res. 2001 Oct 1;61(19):7034-8. states "RASSF1A and RASSF1B transcripts were not expressed in 60% (9 of 15) and 33% (5 of 15) of gastric carcinoma cell lines". I think it is therefore important to run a positive control along the customer's samples. I would recommend to run a HeLa lysate. 2) the customer has no protease inhibitors in the lysis buffer, other than PMSF. This is not sufficient and therefore does not prevent enzymatic degradation of the protein. Please add a cocktail of inhibitors (sigma or Roche for example) to the buffer. The protein can be located in the nucleus so I recommend to use a stronger lysis buffer than a Tris buffer: please use a RIPA or NP40 buffer. (recipes for those buffers and detailed inhibitors can be found on our protocols page https://www.abcam.com/index.html?pageconfig=popular_protocols) Other small points to note are: - the customer mentions "after aliquoting store at 4C"; after aliquoting the antibody should be stored at -20C. Aliquots should be used straight after defrosting and discarded if not used but not kept at 4C for future uses. -the customer mentions lysates being heated for 10min. Can I please make sure this is in reducing loading buffer? 5 minutes at 95C is sufficient. Please make sure the gel is also reducing and check the transfer is adequate. -the customer does not mention what type of secondary is used. I expect it is an anti mouse HRP conjugated product. I recommend to check that it works well with other mouse primary antibodies. -a 12.5% gel would be more suitable than a 10% gel to resolve the protein better. -We find that blocking 1hr in BSA 5% can give better results than milk. Milk, if added in the antibody dilution buffer can also prevent the antibody from binding so if the customer adds milk to the antibody dilution buffer he should try without. Please make sure Tween 20 (0.1%) is present in the TBST antibody and wash buffers. I hope the above recommendations will resolve the customer's problems. Please let me know how he gets on and if he needs further assistance,

I have received feedback today from the laboratory that tested ab23950 and was informed that they have used A431 cells fixed with 4% PFA, methanol has not been tested. Cells were labeled with ab23950 and detection was achieved using a biotinylated secondary antibody and Texas-red conjugated streptavidin. In many cases, the results of staining showed speckled pattern or weak staining in cytoplasm but we couldn’t observe microtubule stained with this antibody. In mitotic cell, we could observe the mitotic spindles stained with this antibody and so thought it was kinetochore stained specifically. The laboratory confirmed that the rest of your protocol looks very similar to yours and believes 4% PFA fixing is most suitable for this antibody. Please find on the datasheet the image of the staining, I hope the above information and image will help,

Thank you for your enquiry. Following some epitope mapping by the lab I can confirm that Mouse monoclonal [3F3] to RASSF1a (ab23950) recognizes specifically the C1 domain(52-101aa) of RASSF1a. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

I have just received feedback from the source of ab23950 and have been informed that we are studying ab23950 with two university groups. Those groups used cell lysate of HeLa as positive control and 293cells transfected with HA-tagged RASSF 1A and RASSF 1C gene. In both groups, there was no problem in detecting a specific band in transfected cells. We are still in the process of re-confirming that the current batch works with HeLa cell lysate as positive control to make sure that the antibody is specific for exogeneous RASSF1a and for endogenous RASSF1a. We have of course previous QC data of this antibody where it detected a band with HeLa and tissues from mouse with no non-specific bands. I apologise for the delay in re-testing this antibody and can offer you a credit note or refund or replacement if you purchased the antibody in the last 90days,

Thank you for your patience and I'm sorry to hear that your customer is experiencing difficulty with this antibody. Our source for ab23950 was able to provide the following details regarding its characterization in Western blotting. The extracts of HeLa cells, mouse brain, mouse liver and mouse kidney tissues (each 20 ug) were resolved by SDS-PAGE, transferred to PVDF membrane and probed with anti-human RASSF1A (1:1000). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a ECL detection system. 293 cell transfected with the HA-RASSF1a gene was also used as positive control. Your customer didn't mention what dilution they tried, but I would suggest starting with 1:1000 with incubation overnight at 4C. Also, I would suggest loading 20-30 ug protein lysate (you customer mentioned 10 ug), and make sure to run a secondary-only control (omit the primary antibody) to ensure that the secondary antibody is not binding non-specifically. I hope this helps. If you need additional assistance, please contact us again.