Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)

Reviews (2)Q&A (10)References (20)

Overview

  • Product name
    Anti-RANKL antibody [12A668] - BSA and Azide free
    See all RANKL primary antibodies
  • Description
    Mouse monoclonal [12A668] to RANKL - BSA and Azide free
  • Host species
    Mouse
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-Pmore details
    Unsuitable for: Flow Cyt
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Recombinant full length protein corresponding to Mouse RANKL aa 1-317.
    Database link: O14788

  • Positive control
    • IHC-P: Human lymph node and liver tissue. ICC/IF: HeLa (human epithelial cell line from cervix adenocarcinoma) cells. WB: Human lymph node lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab45039 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
ICC/IF Use a concentration of 10 µg/ml.
WB Use a concentration of 1 - 2 µg/ml. Detects a band of approximately 35 kDa (predicted molecular weight: 35 kDa).
IHC-P Use a concentration of 1 - 5 µg/ml.
  • Application notes
    Is unsuitable for Flow Cyt.

    • Target

    • Function
      Cytokine that binds to TNFRSF11B/OPG and to TNFRSF11A/RANK. Osteoclast differentiation and activation factor. Augments the ability of dendritic cells to stimulate naive T-cell proliferation. May be an important regulator of interactions between T-cells and dendritic cells and may play a role in the regulation of the T-cell-dependent immune response. May also play an important role in enhanced bone-resorption in humoral hypercalcemia of malignancy.
    • Tissue specificity
      Highest in the peripheral lymph nodes, weak in spleen, peripheral blood Leukocytes, bone marrow, heart, placenta, skeletal muscle, stomach and thyroid.
    • Involvement in disease
      Defects in TNFSF11 are the cause of osteopetrosis autosomal recessive type 2 (OPTB2) [MIM:259710]; also known as osteoclast-poor osteopetrosis. Osteopetrosis is a rare genetic disease characterized by abnormally dense bone, due to defective resorption of immature bone. The disorder occurs in two forms: a severe autosomal recessive form occurring in utero, infancy, or childhood, and a benign autosomal dominant form occurring in adolescence or adulthood. Autosomal recessive osteopetrosis is usually associated with normal or elevated amount of non-functional osteoclasts. OPTB2 is characterized by paucity of osteoclasts, suggesting a molecular defect in osteoclast development.
    • Sequence similarities
      Belongs to the tumor necrosis factor family.
    • Post-translational
      modifications
      The soluble form of isoform 1 derives from the membrane form by proteolytic processing (By similarity). The cleavage may be catalyzed by ADAM17.
    • Cellular localization
      Cytoplasm; Secreted and Cell membrane.
    • Information by UniProt
    • Database links
      • Alternative names
        • CD254 antibody
        • hRANKL2 antibody
        • ODF antibody
        • OPGL antibody
        • OPTB2 antibody
        • Osteoclast differentiation factor antibody
        • Osteoprotegerin ligand antibody
        • RANKL antibody
        • Receptor activator of nuclear factor kappa B ligand antibody
        • Receptor activator of nuclear factor kappa-B ligand antibody
        • sOdf antibody
        • TNF related activation induced cytokine antibody
        • TNF-related activation-induced cytokine antibody
        • TNF11_HUMAN antibody
        • TNFSF 11 antibody
        • Tnfsf11 antibody
        • TRANCE antibody
        • Tumor necrosis factor (ligand) superfamily member 11 antibody
        • Tumor necrosis factor ligand superfamily member 11 antibody
        • Tumor necrosis factor ligand superfamily member 11, soluble form antibody
        see all

      Images

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)

        Paraffin embedded human lymph node tissue stained for RANKL with ab45039 at 5 µg/ml.

      • Immunocytochemistry/ Immunofluorescence - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)
        Immunocytochemistry/ Immunofluorescence - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)

        ICC/IF image of ab45039 stained HeLa cells. The cells were 4% formaldehyde fixed (10 minutes) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab45039, 10 µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

      • Western blot - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)
        Western blot - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)
        Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039) at 2 µg + transfected cell lysate

        Predicted band size: 35 kDa
        Observed band size: 35 kDa

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)

        Paraffin embedded human liver tissue stained for RANKL with ab45039 at a 1/500 dilution. Peroxidase-conjugate and DAB chromogen. A 2 hour incubation at RT was used.

      • Western blot - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)
        Western blot - Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039)
        Anti-RANKL antibody [12A668] - BSA and Azide free (ab45039) at 0.5 µg/ml + human lymph node lysate in RIPA buffer at 35 µg

        Predicted band size: 35 kDa
        Additional bands at: 28 kDa (possible isoform)



        Band detected at ~35kDa and ~28kDa. (Expected MW of 35.5kDa according to NP_003692.1 and of 27.7kDa according to NP_143026.1)

      References

      This product has been referenced in:
      • Ji B  et al. Isoliquiritigenin blunts osteoarthritis by inhibition of bone resorption and angiogenesis in subchondral bone. Sci Rep 8:1721 (2018). Read more (PubMed: 29379010) »
      • Ding LZ  et al. Mangiferin inhibits apoptosis and oxidative stress via BMP2/Smad-1 signaling in dexamethasone-induced MC3T3-E1 cells. Int J Mol Med 41:2517-2526 (2018). Read more (PubMed: 29484386) »
      See all 20 Publications for this product

      Publishing research using ab45039? Please let us know so that we can cite the reference in this datasheet.

      Customer reviews and Q&As

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      1-10 of 12 Abreviews or Q&A

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-RANKL antibody [12A668] - BSA and Azide free

       Excellent
      Abreviews
      abreview image
      Application
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
      Sample
      Rat Tissue sections (mandible)
      Antigen retrieval step
      Heat mediated - Buffer/Enzyme Used: Buffer solutions (Citrate pH 6,0)
      Permeabilization
      No
      Specification
      mandible
      Blocking step
      Hydrogen Peroxide as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C
      Fixative
      Paraformaldehyde
      Read More

      Abcam user community

      Verified customer

      Submitted Aug 30 2016

      Western blot abreview for Anti-RANKL antibody [12A668]

       Good
      Abreviews
      Abcam has not validated the combination of species/application used in this Abreview.
      Application
      Western blot
      Sample
      Rabbit Tissue lysate - whole (Subchondral bone)
      Gel Running Conditions
      Reduced Denaturing (Gel 15%)
      Loading amount
      30 µg
      Specification
      Subchondral bone
      Blocking step
      Milk 5%+ BSA 3% as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
      Read More

      Abcam user community

      Verified customer

      Submitted Jul 01 2016

      Question

      Dear Supplier,

      Please see customers inquiry below:

      For the following antibodies:


      RUNX2 Polyclonal Ab Cat#: ab102712

      RANKL Monoclonal Ab Cat#:ab45039

      RANK Monoclonal Ab Cat#: ab13918

      BMP-7 Polyclonal Ab Cat#:ab56023

      Osteoprotegerin (OPG) Polyclonal Ab Cat#:ab73400

      They are all compatible with IHC. Can you tell me if they will also work for FACS (Runx2; BMP-7 and OPG)?

      Looking forward for your response.

      Thanks

      Kind regards,

      Read More
      Answer

      Thank you for your enquiry.

      All tested applications and species covered by our 6 month guarantee are specified on our datasheets, and these are updated as soon as any new information is brought to our attention.

      Therefore, I can confirm that RANK antibody ab13918 is indeed tested and covered by the guarantee for both IHC-P and flow cytometry.

      RUNX2 antibody ab102712
      This is tested for IHC-P (paraffin embedded sections) as stated on the datasheet. However, I am sorry to confirm this has not been tested in flow cytometry.

      The following RUNX2 antibody is tested in flow cytometry, I hope this may be of interest to your customer. However, please note this has not been tested in IHC and you will also need to check which species the customer is using. This has been tested and covered by the guarantee for human samples only.

      ab135674 RUNX2 antibody
      Tested in: Flow Cyt, WB
      Reacts with: Human
      https://www.abcam.com/index.html?datasheet=135674 (or use the following: https://www.abcam.com/index.html?datasheet=135674).


      RANKL antibody ab45039
      This is tested for IHC-P (paraffin embedded sections) as stated on the datasheet. However, I am sorry to confirm this has not been tested in flow cytometry.

      We do have the following RANKL antibodies tested in flow cytometry. Again, please note that these are not tested in IHC and please also check the species the customer is using:

      ab12125 RANKL antibody [12A380]
      Tested in: Flow Cyt, WB
      Reacts with: Human, Mouse
      https://www.abcam.com/index.html?datasheet=12125 (or use the following: https://www.abcam.com/index.html?datasheet=12125).

      ab95720 RANKL antibody [IK22/5] (Biotin)
      Tested in: Flow Cyt
      Reacts with: Mouse
      https://www.abcam.com/index.html?datasheet=95720 (or use the following: https://www.abcam.com/index.html?datasheet=95720).

      ab93553 RANKL antibody [IK22/5] (Phycoerythrin)
      Tested in: Flow Cyt
      Reacts with: Mouse
      https://www.abcam.com/index.html?datasheet=93553 (or use the following: https://www.abcam.com/index.html?datasheet=93553).

      BMP-7 antibody ab56023
      This is tested for IHC-P (paraffin embedded sections). However, I am sorry to confirm this has not been tested in flow cytometry.

      We do have the following BMP-7 antibody tested in flow cytometry. Again, please note that this is unfortunately not tested in IHC and please also check the species the customer is using:

      ab84030 BMP7 antibody (Biotin)
      Tested in: ELISA, Flow Cyt, sELISA, WB
      Reacts with: Human, Chinese Hamster
      https://www.abcam.com/index.html?datasheet=84030 (or use the following: https://www.abcam.com/index.html?datasheet=84030).

      Osteoprotegerin (OPG) antibody ab73400
      This is tested for IHC-P (paraffin embedded sections). However, I am sorry to confirm this has not been tested in flow cytometry.

      The following Osteoprotegerin antibody is tested in both IHC-P and flow cytometry. Please also check the species the customer is using to ensure it is suitable:

      ab105935 Osteoprotegerin antibody [ 5G2]
      Tested in: ELISA, Flow Cyt, ICC, ICC/IF, IHC-P, WB
      Reacts with: Human
      https://www.abcam.com/index.html?datasheet=105935 (or use the following: https://www.abcam.com/index.html?datasheet=105935).

      If your customer would like to test any of these antibodies in a previously untested species or application, please do not hesitate to contact us again prior to the purchase by replying to this message as you may be eligible for our testing discount program.

      Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

      To find out more about our Abreview system, please see the following link:

      https://www.abcam.com/abreviews

      I hope this information is helpful. Please do not hesitate to contact me for any further advice or information.

      Read More

      Question

      Please could you let us know when we should receive the antibodies in substitution?   Thank you very much

      Read More
      Answer

      I have received confirmation that these 3 replacement vials will be included in the next shipment to Aurogene. I hope this is helpful. Please contact me again if you have any further questions.

      Read More

      Question

      our customer told us that a control can not solve her problems. It could be useful only for the difference between the expected and observed molecular weight (in our case relative to RANKL antibody), but also in that case she's sure that there was no problem in gel running and the molecular weights were indeed those identified by the standard, because after revealing RANKL they have also revealed actin, with the band showing the expected length. Moreover, your the use of a control does not resolve the problem of OPG and ATF4 aspecificity. For this reason, she has requested the substitution of items ab10962, ab23760, ab45039. Please could you let us know when you should ship us these antibodies in substitution? Our customer needs them urgently.  

      Read More
      Answer

      Thanks for your reply. It seems that, as part of their own internal QC, the customer has already peformed the control I suggested. This is great as it does appear clear now that these 3 antibodies are not working as expected. I would be happy to request the replacement antibodies the customer asked for: 1 vial each of ab10962, ab23760, ab45039. So I can process the request, please let me know the purchase order number or Abcam order reference number associated with the initial order of these 3 antibodies. Thanks!

      Read More

      Question

      Hi, I need the antibody to work in human, rat and mouse and for flow cytometry and immunohistochemistry. Regards  

      Read More
      Answer

      Thank you for your reply. I am pleased to provide the following information: We have two RANK antibodies tested flow cytometry, human and mouse (not IHC or rat): ab45092 RANK antibody [9A725] (Phycoerythrin) Mouse monoclonal Tested applications: Flow Cyt Reacts with: Human, Mouse  Click here (or use the following: https://www.abcam.com/index.html?datasheet=45092). ab13918 RANK antibody [64C1385] Mouse monoclonal Applications: Flow Cyt, IP, WB Reacts with: Human, Mouse  Click here (or use the following: https://www.abcam.com/index.html?datasheet=13918). One antibody tested in IHC-P, mouse and human (not rat or flow cytometry):   ab45039 Anti-RANKL antibody [12A668] Mouse monoclonal Tested applications: ICC/IF, IHC-P, WB Reacts with: Human, Mouse Click here (or use the following: https://www.abcam.com/index.html?datasheet=45039). I am sorry to confirm we have no RANK antibodies tested in rat. Regrettably I am not able to find the correct rat sequence on the protein database sites, and so am not able to check the alignments with rat.  If you have access to the rat sequence, I can suggest to try alignment to the immunogen for these antibodies. We recommend alignment should be over 85% to predict that an antibody will detect in a different species. I am sorry we also do not have an antibody tested and guaranteed in both IHC and flow cytometry. If you would like to test one of the antibodies in one of these untested applications, we would be able to provide a testing discount offer. Please let me know if you are interested, and I will be pleased to forward the details. This also applies to the rat species. If the alignment is over 70% , we would be able to provide a testing discount for you to try in rat. I am sorry we do not have an antibody that is tested to meet all your requirements on this occasion, but I hope these ones will still be of interest to you.  If you have any further questions, or are interested in the testing discount, please do not hesitate to contact me.

      Read More

      Question

      LOT NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Non-specific band: I see a band at 55 KDa when I should detect the band at 35 KDa. SAMPLE Soluble cell extract PRIMARY ANTIBODY AB 45039 Human monoclonal anti-RANKL Sample: primary culture human cells dilution 1:500 with 5% MILK 4ºC Over night 3 wash with PBST 0,1% 5 minutes each. DETECTION METHOD Chemiluminiscence (HRP) ANTIBODY STORAGE CONDITIONS At -20ºC in aliquots SAMPLE PREPARATION Fresh RIPA: TRIS pH=8.0, deoxicolic acid, NaCl, NP40 (1%)with protease inhibitors (Complete Mini Protease Inhibitor Cocktail tablet and phosphatase inhibitor 100X). Bromophenol blue (SDS 20%. TRIS 1M pH 6,8, DTT, glycerol, beta.mercaptoethanol) Heating sample at 95ºC during 5 minuts. AMOUNT OF PROTEIN LOADED 40 micrograms ELECTROPHORESIS/GEL CONDITIONS Reducing SDS PAGE gel 10% acrilamide TRANSFER AND BLOCKING CONDITIONS Transfer: 200 mA 2 hours Blocking agent: 5% MILK SECONDARY ANTIBODY Anti-Mouse (HRP) 1:10.000 with 5% MILK 1 hour RT 3 wash with PBST 0,1% 5 minutes each. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

      Read More
      Answer

      Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody. The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. Reviewing this case, I would appreciate if you can confirm some further details to help with our investigations: 1. Please provide the order number reference and date of purchase. If you have ordered through a distributor, they will have this information for you. 2. I would appreciate if you are able to provide an image, including molecular weight markers, which will help us to assess the results. 3. Has a lower dilution of antibody been tried, 1:1000? 4. What type of human cell, which cell lines? Has a negative control been included? 5 5. Please confirm the results of the non primary control In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), I would be pleased to provide a credit note, free of charge replacement or refund. I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested information and details of how you would like to proceed.

      Read More

      Question

      What species of bone cells were being tested?   Murine Osteoblasts from calvaria What dilution of primary antibody for each?   OPG 1:100; ATF4 1:400; RANKL 1:500 Was any other antibody used successfully with these same samples?  No How much protein was loaded on the gel? 20 ug   Please, could you tell us when you'll ship us the antibodies in substitution?  

      Read More
      Answer

      Thank you for providing the additional information. The one thing I would like to recommend in this case is to run a "loading control" western blot with the murine osteoblast cell lysate. Examples of targets for this type of control would be proteins that are commonly expressed at high levels in all cell types, such as actin or GAPDH. This would serve as a control in that detection of the expected band size would indicate that all aspects of the lysate preparation and western blot protocol are working as expected. Thanks so much for your troubleshooting efforts! I look forward to hearing how things go.

      Read More

      Question

      the last June our company Aurogene ordered from you the antibodies ab10962 goat polyclonal to Osteoprotegerin, ab23760 rabbit polyclonal to ATF4 and ab45039 mouse monoclonal |12A668| ro RANKL, that didn't work when used by our customer. In attachment we send you the WB images of these antibodies. The experiments were performed using lysates of bone cells that certainly synthesize proteins for which the above antibodies should be specific. As for the first 2, the customer has found a strong aspecificity, while for RANKL he has seen a single band, but the found molecular weight (65 kDa approximately) is much higher than expected (35 kDa). Please, could you substitute as soon as possible these antibodies?

      Read More
      Answer

       Thank you for your enquiry. Sorry to hear your customer's are having trouble with these antibodies.   What species of bone cells were being tested? What dilution of primary antibody for each? Was any other antibody used successfully with these same samples? How much protein was loaded on the gel? What was the purchase order number or Abcam order reference number associated with these purchases? Thanks in advance for the additional information.

      Read More

      Question

      LOT NUMBER GR24723-3 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Non-specific staining: Non-specific staining: I am using RANKL antibody for IHC in bone marrow from mouse, RANKL antibody is from mouse, then I am using a secundary antibody antimouse from rabbit. I am getting non specific staining. Is RANKL antibody suitable for IHC in mouse? or is it only for human samples? SAMPLE Bone marrow form mouse, I used palstic embedded bone PRIMARY ANTIBODY ab45039, 5 ug/ml, incubation overnight at 4C, 5 wash in PBS 5 changes 5 min with agitation DETECTION METHOD DAB POSITIVE AND NEGATIVE CONTROLS USED Positive control: Skin Negative control: I used bone marrow without primary antibody but with secundary antibody. I used bone marrow without primary or secundary antibody. I got brown staining in the first negative control but not in the secundary negative control. FIXATION OF SAMPLE Methylmethacrylate ANTIGEN RETRIEVAL No PERMEABILIZATION STEP No BLOCKING CONDITIONS 10% horse normal serum, 4% bovine serum albumin SECONDARY ANTIBODY P0161Dako, concentration: 1:200, antimouse, from rabbit, incubation 30 min at room temperature, wash in pbs 5 changes 5 min with agitation HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Primary and secundary antibody concentration. Inhibition of endogenous peroxidase

      Read More
      Answer

      Thank you for your enquiry regarding ab45039 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. I can confirm that anti RANKL antibody is indeed specific to mouse tissue sections. This antibody is fully guaranteed to be used on mouse tissue sections however with additional blocking steps for mouse on mouse use. I have read the protocol you have kindly provided and I have following comments/ suggestions to make; In negative control samples you have mentioned that you have observed a brown staining with no primary antibody control, this means the anti mouse secondary is actually binding to the endogenous mouse IgG which is giving back ground staining. Please find attached pdf file in which additional blocking steps are nicely explained. https://www.abcam.com/index.html?pageconfig=resource&rid=11465 I am sure these suggestions will help to improve your results. Should you have any questions or concerns please do not hesitate to contact me.

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