Anti-Survivin antibody (ab469)

Good afternoon, Thank you for your fast response to my e-mail. First aswering to the last two questions: I've been using Streptavidin protein (HRP) (ab7403) as for thedilution of the secondary antibody is 1/250 Nowwith regard to the form sent: 1) Abcam product code ab469 3) Description of the problem: Need more intense staining 4) Sample preparation: Species: Rat Type of sample:PFA/formalin fixed paraffin embedded sections Negative control: Removal of primary antibody 5) Fixation step Yes If yes: Fixative agent and concentration:paraformaldehyde 4% Fixation time: at least 48 hours Fixation temperature 4ºC 6) Antigen retrieval method: Heat-induced with 10mM Sodium Citrate Buffer 7) Permeabilization method: Not done 8) Blocking agent (eg BSA, serum…):Normal Goat serum Concentration 5% in TBST Blocking time: 1h Blocking temperature: room temperature 9) Endogenous peroxidases blocked? Yes (3% Hydrogen Peroxide) Endogenous biotins blocked? Yes (Endogenous avidin + biotin blocking system ab3387) 10) Primary antibody (If more than one was used, describe in “additional notes”) : Anti-Survivin (ab469) Concentration or dilution 1/500 Diluent buffer1xTBST Incubation time Overnight 11) Secondary antibody: Goat Polyclonal Secondary antibody to Rabbit IgG - H&L (Biotin) (ab6720) Concentration or dilution 1/250 Diluent buffer1xTBST Incubation time 2h 12) Washing after primary and secondary antibodies: Yes Buffer: 1x TBST Number of washes at least 3 times 13) Detection method: Streptavidin Protein (HRP) (ab7403) DAB substrate kit (ab94665) 14) How many times have you run this staining? Several Times Do you obtain the same results every time?yes almost every time What steps have you altered to try and optimize the use of this antibody? Altering the dilution of primary antibody, altering the time of incubation of the primary antibody, altering the time of incubation of the cromogen. Best regards,

ANSWER:

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: http://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.

Good morning, I'm currently using Abcam products to perform imunohistochemistry on paraffinon my research protocols and I have some doubts that I would likeAbcam tohelp me. I worked with Anti-Survivin antibody (ab469) as a primary antibody in a 1/500 dilution. I useda goat polyclonal secondary antibody (ab6720) as well as endogenous Avidin + Biotin Blocking System (ab3387) and DAB Substrate (ab94665)kit as a cromogen. With this experience Iwas able to get favorable results, however I would like to know how to get a more intense staining?I tried to optimize the dilution of primary antibody as well as the exposure time of DAB butcould not get significant changes. Could you give mean advise on how to get some more intense staining? Another question about the primary antibody Anti-Smac / Diablo (ab8114) is what dilution do you recomend using in IHC-P protocols? I read the datasheet where you say to use a concentration of 5ug/ml but I can't understand how to perform this concentration/ diluition. Best regards,

ANSWER:

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.

We are using your rabbit polyclonal anti-survivin antibody (ab469).  According to the data sheet, the immunogen is "recombinant full length protein."  Do you know whether this anitbody is, therefore, specific only for the Wild Type, or does it also recognize other splice variants?  

ANSWER:

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.

 Thanks for your help. The membranes were blotted with the GFP Ab and then stripped and re-probed with Ab-survivin. The gel on the right is in the absence of FBS (fetal bovine serum). I didn’t try a different GFP Ab. The fact that the anti-GFP pic a 17bp band in the cells overexpressing GFP only makes me think that band is not specific. Do you recommend a better surviving Ab?    

ANSWER:

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: http://www.abcam.com/index.html?datasheet=24479).  

 The Ab Lot number is ****. We use 5%milk to block the membrane and I tried boiled and not boiled samples. Please see attached ppt for the westerns done with different Abs. NT stands for not transfected. What loading buffer do you advise to enrich the monomer and get rid of the dimer.    

ANSWER:

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.