Overview

  • Product name
  • Description
    Rabbit polyclonal to BMP2
  • Host species
    Rabbit
  • Specificity
    ab14933 detects only the pro-form of the BMP2
  • Tested applications
    Suitable for: Dot blot, ELISA, WB, ICC/IF, IHC-Fr, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Mouse BMP2 aa 45-60 (N terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
    Sequence:

    SEDVLSEFELRLLSMF


    Database link: P21274
    (Peptide available as ab80798)

Properties

Applications

Our Abpromise guarantee covers the use of ab14933 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
Dot blot 1/2000.
ELISA 1/2000.
WB 1/500 - 1/1000. Detects a band of approximately 45 kDa.Can be blocked with Mouse BMP2 peptide (ab80798).
ICC/IF Use a concentration of 1 µg/ml.
IHC-Fr 1/250 - 1/500.
IHC-P Use at an assay dependent concentration.

Target

Images

  • Anti-BMP2 antibody (ab14933) at 1/500 dilution + BMP2 Purified Protein
  • ab14933 at 1/500 dilution staining human bone tissue sections by Immunohistochemistry (Frozen sections). The tissue was fixed with methanol and blocked with BSA prior to incubation with the antibody for 14 hours. StreptABComplex/HRP duet was used as the secondary antibody in this experiment. In the image specific staining is shown in brown at the site of new bone formation. The blue color is due to toluidine background staining.

    See Abreview

  • ICC/IF image of Rat primary hepatocytes culture cells stained with ab14933. The cells were fixed with 4% PFA.and incubated in  5% normal donkey serum in 0.1% PBS- and triton X100 for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14933, 1µg/ml) and (ab15640) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 568 (red) at a 1/1000 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
  • ab14933 at 1/200 dilution staining BMP2 in human 293FT cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde and blocked in 5% BSA for 1 hour at 25°C. The primary antibody was used at 1/200 dilution in PBS and incubated with sample at 4°C for 12 hours. An Alexa Fluor® 488 conjugated Goat polyclonal to rabbit IgG was used as secondary at 1/200 dilution.Cells were cultured in DMEM containing 10% FBS and Nuclei stained with DAPI.

    See Abreview

References

This product has been referenced in:
See all 41 Publications for this product

Customer reviews and Q&As

1-10 of 30 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Cartilage)
Antigen retrieval step
Enzymatic - Buffer/Enzyme Used: Proteinase K 100ug/ml
Permeabilization
No
Specification
Cartilage
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1.5% · Temperature: 24°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Dec 01 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (aortic smooth muscle)
Gel Running Conditions
Non-reduced Denaturing
Loading amount
20 µg
Specification
aortic smooth muscle
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted May 06 2016

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Sample
Human Cell (MCF7 cells)
Specification
MCF7 cells
Permeabilization
Yes - 0.25% Triton, 15 min
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jan 07 2015

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (MCF-7 cells)
Specification
MCF-7 cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 07 2015

Answer


For the detection of BMP2 in IHC I would recommend the Rabbit polyclonal ab14933 which is guaranteed to work in both IHC-Fr and IHC-P as well as on Mouse, Rat and Human samples. The link to the datasheet is www.abcam.com/ab14933.
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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Answer

Thank you for your enquiry and your interest.

We have not specifically tested this but as the immunogen for ab82511 (and ab14933) is designed to aa 45-60 and Swiss-Prot states the location of the amino acids that are glycosylated are at aa 135, 163, 164 and 200.

It seems likely that ab82511 should recognize both the glycosylated and non glycosylated immature protein. This ab will not recognize the non-glycosylated mature protein (aa 283-396) which you may wish to do.

We did test ab17885 with mature recombinant protein (ab87065) last year and that showed a really nice band at the expected MW of ˜14 kDa. Though, we are not sure if it has the species cross-reactivity you are looking for.

I hope this helps and if I can assist further, please do not hesitate to contact me.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1124943.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

Thank you very for contacting us.

This antibody is one of the high seller product and we haven't received many specificity complaint for this product so we are unsure why this particular vial is not working with your customer. I have few further recommendations that might help to improve results;

- In our own lab we test both milk and BSA as blocking solution. 5% BSA, 2 hours incubation time is the most common and gives excellent results.
- In our lab we heat the lysates for 10 minutes at 100C.
- No primary control could also help to check the non specificity of secondary antibody
- The problem might with lysates (isolation of fibroblast or lysis etc.), I can recommend using lung, spleen and colon tissue lysates as positive control.

I can also offer your customer a replacement vial from different lot for ruling out completely, the problem due to antibody itself.

Please let me know how the customer would like to proceed further.

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Answer

Thank you for providing details quickly.

The protocol seems absolutely fine. The antibody should have worked, however we are not sure why this antibody is binding non specifically. In order to know the cause of multiple bands, is it possible for you to run test with human lysates? This could be a good positive control to check if the problem is due to antibody or preparation of lystaes.

Let me know what you think?

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Question

Product code: 14933
Lot number: GR41047-2
Inquiry: OUR PO WAS: 543 1)Antibody storage conditions:Reconstitued antiboby has been store at –20°C. 2)Description of the problem: More bands 3)Sample: Protein have been extrated from mice dermal fibroblast and it has been done a total lysis 4)Sample preparation:Cells were washed several times with phosphate-buffered saline and homogenized in RIPA buffer in presence of protease inhibitors. Cellular lysates were centrifuged at 15,000 rpm for 20 min to clear cell debris, and supernatants were collected and stored at –80°C until analysis. 5)Amount of protein loaded: 30 µg proteins 6)Electrophoresis/Gel conditions: 10% polyacrylamide gel, under reducing conditions 7)Transfer and blocking conditions:Transferred to nitrocellulose. Membrane was blocked in TBS+0.1% Tween 20 + 5% non fat dry milk for 1 hr at room temperature 8)Primary Antibody:Antibody was diluted in TBS-0.1% Tween 20 + 2.5% non-fat dry milk at 1:1000 and incubation time was overnight at 4°C. 3 wash steeps and incubation of nitrocellulose with 9)Secondary Antibody:Secondary antibody (no Abcam antibody) was diluted in TBS-Tween 20 + 2.5% non-fat dry milk at 1:5000 for 1 hr. 10)Detection method: Western blots were visualized using Super Signal West Pico 11)Positive and negative controls used (please specify):I used fibroblast isolated from human skin and in this case I used an other antiboby (bought by an another company) that recognized only a band. 12)How many times have you tried the Western?: 3 times 13)Have you run a "No Primary" control?:No 14)Do you obtain the same results every time?: Yes and the number of bandes were always the same. 15)What steps have you altered?: I modified concentration of antibody both primary that secondary and the incubation time (instead overnight I tried only 1hr)

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Answer

Thank you for contacting us. I am sorry to hear that you are experiencing problems with this antibody.

I have checked the protocol and would like to ask few further questions for better understanding of the problem;

- "Reconstituted antibody has been store at –20°C", could you explain what this means? Did you get the product lyophilized or you just mean diluted the antibody and stored?
- Could you send us an image?
- You have said you have tried antibody from different company and it worked. Was it with the same protocol and lysates?

I look forward to hearing from you soon.

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1-10 of 30 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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