Anti-Survivin 抗体 (ab469)

Thank you for providing that extra information. It has helped greatly to understand what you have been doing and what may help to improve the signal of the staining. There are a few areas that I would suggest would be worth optimising (if you have not already done so) which may lead to an improved signal. 1. Fixation You have stated that you are fixing the tissue for a minimum of 48 hours. The ideal fixation time will depend on the size of the tissue block and type of tissue but usually 18-24 hours seems ideal for most applications. Over-fixation can lead to the epitope being masked. 2. Antigen retrieval Antigen retrieval can be used to overcome the masking mentioned for the fixation. Depending on the level of masking, different conditions of antigen retrieval may be required. For example, if using a microwave we would usually suggest trying retrieval for 5, 10, 15 and 20 minutes to see which produces the optimal results. 3. Hydrogen peroxide blocking I would suggest performing the hydrogen peroxide blocking following the incubation of the primary antibody if you have not already tried this. The hydrogen peroxide can sometimes affect sensitive epitopes and by performing the step following the incubation with the primary antibody this will not affect the staining. 4. Primary antibody incubation You mention that you have optimised this step by changing the dilution and incubation time. If you have not already done so I would suggest attempting the incubation for 1-1.5 hours at room temperature with agitation. 5. Detection method Different techniques can be used to directly amplify the signal. Currently you are using streptavidin-HRP conjugate which is labelled in a 1:1 ratio. It is possible to employ avidin which has been labelled in a 3:1 ratio with HRP using kits such as Piercenet product 32020. Although avidin can cause greater background compared to using streptavidin. Alternatively HRP polymer can be employed. This consists of using a secondary antibody which is attached to a polymer-HRP complex. Abcam has the following product to offer, ab2891, however this is a little pricey. I would suggest trying to optimise the steps 1-4 and if sufficient progress is not made contemplate employing one of signal amplification methods mentioned. We have quite a detailed guide to IHC which may be of help to you. This outlines many of the different experimental parameters which need to be considered when performing IHC and suggestions to improve experiments: https://www.abcam.com/ps/pdf/protocols/ihc_p.pdf I hope this information has been of help and your staining improves. If you want any further information or help please do not hesitate to contact us again.

Thank you for contacting us. I'm glad to hear you have been getting good staining with ab469. In order to help you obtain more intense staining would you mind filling in the form which I have attached to this email. It will allow me to more fully understand the protocol which you have been performing and any areas in which it may be worthwhile to optimise. Could you also answer the following questions: 1. which HRP conjugate have you been using (streptavidin-HRP? could you tell me the catalogue no. etc) 2. in what dilution have you been using the secondary antibody? ab8114 is supplied at a concentration of 1 mg/mL. The recommended working concentration of this antibody when using it for immunohistochemistry with paraffin embedded sections is 5 µg/ml. This equates to a dilution of 1/200. This is only a guideline and may need to be optimised for the staining which you are performing. I hope this information has been of help and look forward to your reply.

Thank you for contacting us. A similar question was asked by a customer in May of 2008. The question, which contains some references, and our reply, can be found under the Scientific Support tab of the online datasheet. Without testing reactivity with the splice variants, we cannot be sure which the antibody will detect. However, the splice variants listed in the UniProt database entry for the human survivin, accession O15392, (the URL is http://www.uniprot.org/uniprot/O15392) all share the same 73 N-terminal amino acids. Given that this antibody is raised against the full-length isoform 1, also know as alpha isoform, which inludes the 73-amino acid range, and assuming that the antibody recognizes at least one epitope in that range, the antibody should be capapble of recognizing all splice forms. However, depending on how the protein folds, epitopes in the N-terminal range may be inaccesible to the antibody. This should be considered a possibilty if you are trying to detect survivin in its native conformation, for instance by IHC or ELISA. The ability to make a consistent prediction of reactivity with variants is also complicated by the nature of polyclonal antibodies, insofar as the epitopes they recogize will vary with each immunization and the resulting sera. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you the clarification. The 17 kDa band is strange, insofar as it appears in the GFP-stained blot, looks very similar to the `17 kDa band in the survivn-stained blot, and appears only in the lanes containing transfected cell lysates, which argues against it being endogenous survivin. I think you may be getting cleavage of the survivin from the GFP. That would explain the 17 kDa band in the lanes of transfected samples in the survivin blot. I am still having trouble understanding the differences among your samples. In particular, can you tell me how YFP-CON differs from YFP-SURV? I am assuming that YFP-CON is just a transfection with YFP, based on the GFP signal in those lanes. But then there is no explanation for the 17 kDa survivin signal in those lanes, in the survivin blot. For an alternative survivin antibody, I suggest ab24479, which gives clean blots on a variety of samples. Click here (or use the following: https://www.abcam.com/index.html?datasheet=24479).  

Thank you for sending the images. The blots stained with ab469 have an isolated band at 17 kDa which I assume is endogenous survivin, but that same band appears in the GFP-stained blots. Were the anti-GFP blots also stained with ab469? Also, what is the difference between the blots on the left from the ones on the right, in the ppt file you sent? What is the sample on the far right in the left-side blots? The images you send do make the problem more clear, though, than the earlier TIFF file. I do not think multimers are the problem, assuming you are adding a reducing agent in your loading buffer before boiling the sampes. What is interesteing is that the GFP antibody (if it is the only one being used to stain the upper blots) is picking up the same multiple bands as the survivin antibody, suggesting that your cells may be producing several truncated transcripts of different sizes. I cannot tell if the bands are the same size though, since the markers in the survivin blots are not labeled. Have you tried any other GFP antibodies? If you see the same pattern, then you may need to consider that possibility of multiple transcripts. I look forward to your reply, and to resolving this issue. It may require another antibody.

Thank you for contacting us about the western blot issue you are having with this antibody. We typically ask for a few details of the protocol to see if there are modifications we can suggest, but I expect you have a standard protocol that works well with other antibodies. We would also like to have the lot number of the antibody. It will be on the tube. If you do not have that, can you tell me the PO number or approximate date of the order? Some of the bands in the blot image appear to be multimers of the 16 kDa survivin monomer, along with other bands that may represent non-survivin proteins that the antibody cross-reacts with. We have not received similar reports. Can you please tell me what the samples are and how they are prepared for loading into the gel? What do you use for blocking the membrane, and is the secondary effective with other rabbit antibodies, with these samples? It is unlikely that the secondary is at fault but we ask, just in case it is a new antibody in the lab. Finally, which lanes contain the GFP-survivin, and which contain the un-transfected cell lysates? I look forward to your reply. If I cannot offer a suggestion, we will replace the antibody with a different lot or a different antibody altogether. If you prefer, we will also consider a credit or refund. I look forward to your reply.