Overview

  • Product name
    Anti-Bone Sialoprotein antibody
    See all Bone Sialoprotein primary antibodies
  • Description
    Rabbit polyclonal to Bone Sialoprotein
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ELISA, RIA, WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Rat, Human
  • Immunogen

    Human Bone Sialoprotein (bone extract)

Properties

Applications

Our Abpromise guarantee covers the use of ab52128 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ELISA Use at an assay dependent concentration.
RIA Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 35 kDa.
IHC-P Use at an assay dependent concentration.
ICC/IF 1/100.

Target

  • Function
    Binds tightly to hydroxyapatite. Appears to form an integral part of the mineralized matrix. Probably important to cell-matrix interaction. Promotes Arg-Gly-Asp-dependent cell attachment.
  • Post-translational
    modifications
    N-glycosylated; glycans consist of sialated and core-fucosylated bi-, tri- and tetraantennary chains.
    O-glycosylated at eight sites; mucin-type glycans contain Gal, GlcNAc, GalNAc and terminal NeuAc.
    Sulfated on either Tyr-313 or Tyr-314.
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • BNSP antibody
    • Bone sialoprotein 2 antibody
    • Bone sialoprotein II antibody
    • BSP antibody
    • BSP II antibody
    • BSPII antibody
    • Cell binding sialoprotein antibody
    • Cell-binding sialoprotein antibody
    • IBSP antibody
    • Integrin binding sialoprotein antibody
    • Integrin-binding sialoprotein antibody
    • SIAL_HUMAN antibody
    • SPII antibody
    see all

Images

  • ab52128 (2ug/ml) staining Bone Sialoprotein in human bone using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of Bone sialoprotein in sectretory compartments in the cells of the perichondrium
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

References

This product has been referenced in:
  • Bianco D  et al. Alterations of Subchondral Bone Progenitor Cells in Human Knee and Hip Osteoarthritis Lead to a Bone Sclerosis Phenotype. Int J Mol Sci 19:N/A (2018). Read more (PubMed: 29415458) »
  • Liang D  et al. Effects of nicotine on the metabolism and gene expression profile of Sprague-Dawley rat primary osteoblasts. Mol Med Rep 17:8269-8281 (2018). Read more (PubMed: 29658611) »
See all 11 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Thank you very much for your reply.

The replacement vial of ab125227 will ship out with our next delivery to Biogen. Due to Customs restrictions, this replacement will ship out at the full price ($303.30) but I have arranged credit note # **** to reimburse you for the price.

Please keep me updated about how this replacement antibody works for the customer, and let me know if you have any questions or need anything else from us.

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Question

We did the test accordingly to your instructions:

Antigen Retrieval: Pre-heated Citrate Buffer pH 6,0 (95-99oC). Water-Bath for 30 minutes.

Peroxidase Blocking: Hydrogen Peroxide Block Reagent (Spring – Code DHP-125), for 20 minutes, at room temperature (20-25oC).

Protein Blocking: Protein Block Reagent (Spring - Code DBP-125), for 10 minutes, at room temperature.

Primary Antibody: Diluted 1:500 in Antibody Diluent (Spring - Code ADS-125). Overnight Incubation (16 hours), at 4oC.

Detection System: Polymer-HRP N-Histofine Simple Stain Rat Tissue MAX PO (MULTI) (Nichirei - Code 414191F), for 30 minutes, at room temperature.

Chromogen: Liquid DAB (Spring - Code DAB-125), for 10 minutes, at room temperature.

But, unfortunately, the results weren't good. In this test we could see better the immunostaining, but accordingly to our costumer it's non-specific. I mean, the antibody is staining all the tissue structures, being impossible to identify the bone cells (see attached images 1 and 2). At this test reaction, we also did negative control slides (omitting primary antibody) and they are all OK (none immunostaining observed on these slides), what indicates that the non-specific staining observed on the positive slides (incubated with ab52128) are really caused by the primary antibody.

Our costumer is comparing this new test results with ab52128 with the results obtained with another antibody anti-Bone Sialoprotein (from another manufacturer) she had used before (see attached images 3 and 4). With this other antibody we can see that some structures on the tissue are stained and others no as expected, but the same wasn't observed for ab52128 (compare with images 1 and 2).

What do you think about that case?

Do you have any other suggestion for trying to improve protocol?

I'm forward to hearing from you

Thanks for your attention.

Read More
Answer

Thank you very much for your reply.

I am sorry to hear that the results are still not satisfactory, and I appreciate how much time the customer has spenttroubleshooting. It sounds like the antibody is not work as expected, and I would be happy to send a replacement or issue a credit or refund if the customer would prefer. We have a different bone sialoprotein antibody that is guaranteed to work with rat samples in IHC-P, which I can send as a free of charge replacement to the customer-

https://www.abcam.com/Bone-Sialoprotein-antibody-ab125227.html

Please let me know if the customer would like to accept one of these options, and I will arrange it for you promptly. I look forward to hearing from you, and if there is anything else that we can do please let me know.

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Answer

Thank you very much for your reply and for sending the additional information.

We have not tried the microwave method with this antibody, but one of the other methods (steamer or pressure cooker) may work better. I would suggest using the steamer, but only heating the slides for 20-30 minutes to avoid over-retrieving the tissue. The slides can be allowed to cool for 10-20 minutes on the benchtop.

Please keep me updated about the results after trying the alterations that we've discussed, and if the staining does not improve then I'd be happy to send a replacement or issue a credit or refund. I look forward to hearing from you.

Have a nice day!

Read More

Answer

Thank you very much for contacting us and letting us know about the customer's trouble with this antibody.

Threemonths in EDTA is not too long for decalcification. Bone can be a difficult tissue to work with, soit's possible that there is a problem with thevial but there may also be some protocol alterations that will help.

I've been in touch with the lab regarding this antibody, and we may have a few recommendations that might improve the results. How long was the tissue heated in the citrate buffer for antigen retrieval?

For blocking, we would recommend using 5-10% serum to reduce non-specific binding. An overnight incubation with the primary antibody at a higher dilution (1:500 or 1:1000) may also increase the specific binding of the antibody.

We do fully guarantee this antibody to work in IHC-P with rat tissue, so if the customer is not satisfied with it then we can send a replacement, or issue a credit. Please let me know if the customer would prefer one of these options.

I look forward to hearing from you. If there is anything else that we can do for you, please let me know and I'll be happy to help.

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Endochondral bone)
Specification
Endochondral bone
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 19°C

Abcam user community

Verified customer

Submitted Jan 25 2011

Answer

Thank you for your inquiry. Unfortunatly we do not have a specific dilution to recommend for these antibodies. I can suggest to try a range of dilutions from 1:100 to 1:3000 and to optimize the dilution according to the results. Too much antibody will result in non-specific bands. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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