Anti-Prion蛋白PrP抗体(ab703)

Thank you for contacting us again and for the full and clear explanation of your experiments and results.


I am sorry that the results with this antibody has not been more successful as was indicated by the previous results with this antibody. It does indeed appear that the antibody is not detecting any PrPsc whilst the 8H4 clone is picking it up. The reason for this is unclear. From the following paper it is indicated that the Chandler strain is much more susceptible to degradation than other strains, which may mean that the levels present following PK digestion are low. It also states that the monoglycosylated form is most prevalent in this strain which may expain the single band at ˜25kDa. However, as 8H4 was able to pick out the PrPsc and the single band observed with ab703 is so clean these explanations may be stretching it a bit.


http://www.ncbi.nlm.nih.gov/pubmed/10415165


The conditions used to obtain the Western blot presented on the datasheet of ab703 was to treat the sample with PK (at a concentration of 20 µg/mL) for 30 minutes at 37°C then loaded on the gel following heating to 99°C for 10 minutes. May I ask, how did you quench the PK reaction? The paper above used the addition of PMSF. Were the samples used for the 8H4 and ab703 exactly the same (i.e. had they both been frozen prior to detection)?


I would say from the results obtained it may be most worthwhile to optimise the conditions using the monoclonal antibody 8H4. Whilst the bands are not clear, especially for the PrPc, this may be improved on optimisation. Concentrating on the incubation of the antibody with the membrane as well as the blocking conditions used. It may also be necessary to look into how the samples are prepared, as the protein has a tendency to aggregate which may be contributing to the higher bands observed for the PK- lanes.


For the incubation, I would try both 1 hour at room temperature as well as at 4°C overnight (with agitation). This can quite often lead to a dramatic reduction in non-specific banding. I would also try an alternative blocking agent such as 3% BSA in TBST or 5% serum.


If you would like a replacement with our 8H4 antibody or any of our other anti-PrP antibodies I would be happy to arrange this. I am not sure ab52604 would be an improvement as the specificity has not been assessed and the band observed by Western blotting is again indicating the detection of only one glycosylated form.Alternatively if you would prefer a refund please do let me know.


I am sorry I have not been able to beof more help in this case.

Thank you for contacting us again regarding our antibodies directed against PrP. I am sorry that I was not in the office and able to deal with your call. I am sorry that it has taken some time to find more information relating to the antibodies we currently have to offer and which may be the most suitable for you. We have a number of antibodies which have been confirmed to recognise both the PrPc and PrPsc forms of the protein. These include: ab6664, ab703 and ab14219. I am unsure as to how you are currently performing your experiments but it seems that the normal protocol is to use proteinase K to destroy any PrPc in your sample and then any protein which is left (PrPsc) can be detected using Western blotting. As discussed over the phone, ab703 seems to be appropriate for your experiments, however I am a little concerned that the results that you have been observing have been highly non-specific and the Western blot for this antibody has shown to give quite broad bands. This antibody is covered by the Abpromise and if you do choose to buy it we would offer you all the support we can to make it perform as expected and if you were to be unhappy with the results youmay be entitled to a free of charge replacement or a refund. I would suggest considering ab52604 as this antibody has shown very clean Western blotting bands with human fetal, mouse and ratbrain. I have attached the image to this email. Although this antibody has not been tested to see if both forms of the protein are recognised, it seems likely that it would, using the same procedure of proteinase K treatment. However, as this has not beenconclusivelyconfirmed I would be willing to offer a testing discount to you. This would involve you buying this antibody, testing it with your samples to assess the specificity and once you have shared the results with us through submitting an Abreview of your results (should only take 5-10 minutes) you would be entitled to a primaryantibody of your choice from our catalogue. More information onthis offer can befound here: https://www.abcam.com/collaboratordiscount If you would be interested in this offer please let me know and I can issue you witha discount code. I am still trying togather more informationregarding theotherantibodiesinourcatalogueand will keep youupdated with any furtherrelevant information. I hope this information has been of help.If you require any further information please let me know.

Thanks for the additional information. Here is a link to an HRP-conjugated secondary antibody you could for western blotting with ab703: https://www.abcam.com/Rabbit-IgG-secondary-antibody-ab16284.html I hope this is helpful. Please contact me again if  you have any further questions.

Thank you for your enquiry. I would be happy to help you find a secondary antibody for use with ab703. If you could let me know what application you would use the antibody for and what conjugate you would like attached to the secondary (eg. HRP, biotin, what color fluorophore) then I can recommend a product.

Thank you for your enquiry and your patience. I was away so I could not answer to this technical enquiry sooner. I managed to get some more information about this product: - 12% SDS polyacrylamide gel - 25-50 ug total protein are loaded - rabbit polyclonal to prion protein diluted 1:2000 - Loading buffer is: 150 mM tris-HCl, pH 6,8, 6% SDS, 0.03% bromphenol blue, 30% glycerol - sample incubation 95°C for 5 min - the membran is developed by enhanced chemilumenscence (ECL) .