Overview

  • Product name
  • Description
    Rabbit polyclonal to TEM8
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide:

    LMKLTEDRE QIRQGLE

    , corresponding to amino acids 92-107 of Human TEM8.

  • Positive control
    • WB: mouse kidney lysate IHC: mouse lung, skin and intestine IF/ICC: HepG2 cell line.

Properties

Applications

Our Abpromise guarantee covers the use of ab19387 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
IHC-P 1/100.
ICC/IF Use a concentration of 1 µg/ml.
WB 1/1000. Detects a band of approximately 45 kDa (predicted molecular weight: 63 kDa).

We recommend using 5% BSA as your blocking buffer and antibody diluent.

Target

  • Function
    Plays a role in cell attachment and migration. Interacts with extracellular matrix proteins and with the actin cytoskeleton. Mediates adhesion of cells to type 1 collagen and gelatin, reorganization of the actin cytoskeleton and promotes cell spreading. Plays a role in the angiogenic response of cultured umbilical vein endothelial cells.
  • Tissue specificity
    Detected in umbilical vein endothelial cells (at protein level). Highly expressed in tumor endothelial cells.
  • Involvement in disease
    Defects in ANTXR1 are associated with susceptibility to hemangioma capillary infantile (HCI) [MIM:602089]. HCI are benign, highly proliferative lesions involving aberrant localized growth of capillary endothelium. They are the most common tumor of infancy, occurring in up to 10% of all births. Hemangiomas tend to appear shortly after birth and show rapid neonatal growth for up to 12 months characterized by endothelial hypercellularity and increased numbers of mast cells. This phase is followed by slow involution at a rate of about 10% per year and replacement by fibrofatty stroma.
  • Sequence similarities
    Belongs to the ATR family.
    Contains 1 VWFA domain.
  • Domain
    Binding to PA occurs through the VWA domain.
  • Cellular localization
    Cell membrane. Cell projection > lamellipodium membrane. Cell projection > filopodium membrane. At the membrane of lamellipodia and at the tip of actin-enriched filopodia. Colocalizes with actin at the base of lamellipodia.
  • Information by UniProt
  • Database links
  • Alternative names
    • Anthrax toxin receptor 1 antibody
    • ANTR1_HUMAN antibody
    • Antxr1 antibody
    • ATR antibody
    • Tumor endothelial marker 8 antibody
    see all

Images

  • ICC/IF image of ab19387 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab19387, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
See all 4 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
Answer

Thank you for contacting us. The TEM8 antibody ab19387 was raised against a specific region in the mouse TEM8 that does not show homology to CMG2 (also known as ATR2). 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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Question

find attached a word document with answers. My results do not conclude anything about mouse. I am already trying to understand what might be happening and would be very grateful if you can clarify a few points: -there are several bands on all blots, I'd be very interrested to know their sizes but with only one MW marker am finding it hard to determine this (are the bands above 78kda close to 100kDa for example, or 80kDa?) These are criterion gradient gels from 5 to 15%. I’m not sure which bands you refer to. I’m including at the end of the file a scan with the complete gel FYI. -Looking at the western blots of the lysates, I see one band with the Mono (I guess this is the 80Kda band), but two bands both with the poly and the HA tag antibodies. I am wondering if the HA tag is not also a poly. In general polys do pick up better various forms of the proteins and it could be that those two polys are picking up cleaved forms of TEM8. Could this be the case? In the case of the poly, there are many bands but I can’t comment because none of them looks like the ones recognized by HA or the mono when you compare the MW or the shape. The lower MW band picked up by HA is not specific since it is found in the control lysate too. The band for TEM8 is seen as a doublet because it is a re-probing after using the monoclonal. When you strip blots, you take off the secondary antibody but not the primary, so you see it as an “empty space”. The HA tag antibody is a rat monoclonal and recognizes a tag in the C-terminus of the protein. -Looking at the IPed reprobes, it seems again to me that the poly anti TEM8 and the HA tags are giving similar band patterns, wether it was an IP with HA antibody or the poly. So with the IP with the poly it seems to me that the reprobe with the HA does give something, and as the TEM8 is expressed not endogenously in your cells I am wondering if again it could be because of a cleavage of the protein. Interrestingly IPing with the mono did not give a band at 80kda, so I conclude that the mono does not work well in IP, is this your opinion too? Yes, the mono is good only in western blots. The Ipps suggests that the Abcam antibody does IP Tem8 but what concerns me is that when you IP with Abcam and probe again with Abcam, you don’t see the expected band. I don’t think that it is a problem with Abcam Ab working in Western blots because it recognizes the expected band when Iped with anti HA. I don’t know what are the bands right below but I think it is unspecific because you see it in all 3 lanes with ipps and even in the lysates. I would be very grateful if you can let me know your thoughts on those points so I can better understand what might be happening in your experiments. The MW markers are: 200, 130, 78, 39, 32, 17, 7.

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Answer

Thank you for your reply and providing more information and a whole blot image. Your problem puzzled me greatly and I also asked other colleagues to help understand what might be happening. Here are a few of our thoughts: -For the western blots: you said "The band for TEM8 is seen as a doublet because it is a re-probing after using the monoclonal. When you strip blots, you take off the secondary antibody but not the primary, so you see it as an “empty space”." Have you tried using the antibody on a fresh membrane? Stripping can damage the proteins and therefore prevent them from being detected by the second primary antibody. Furthermore if there is still primary antibody from the first experiment the second primary antibody will have more problems binding to the protein. -The Abcam antibody has not been tested on human samples, and as the protein is expressed in different isoforms/ splice variants we are wondering if the Abcam antibody is better at detecting one splice variant over an other, hence in our samples of mouse kidney it can detect the protein well in the three isoforms while in your samples the expression pattern may be very different. We would recommend to run a positive control of mouse kidney whole cell lysate to determine if this is the case. For the Immunoprecipitations: -The small strong large band in the IPed samples (HA and Poly IPed) and reprobed with the polyclonals could be the 60kDa form of the protein, and the strong large band could be the 45kDa form of the protein. As the poly has not been tested in IP, it's very hard to know if it is working optimally for this application. Again, having a fresh membrane would be useful to see exactly what bands are due to what antibody. As the antibody has not been tested in human samples I think the best recommendation I can make is to run a positive control along your samples; we do not guarantee ab19387 to work in human but I would be more than happy to offer you a refund or another antibody if you still have a problem with the positive control.

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Answer

I'm sorry to hear you are experiencing unsatisfactory results with ab19387. I would be happy to consider offering you a replacement vial of ab21270, however before I can do that I would like to investigate this matter further to make sure that the problem with ab19387 could not be solved with a few protocol modifications. The quality of our products is of utmost importance to us and should we find out that ab19387 is of poor quality we will not hesitate to remove it from the catalogue. Can you please click on the link below which will take you to a short protocol questionnaire to help you put the information together, and could you please put it to my attention so I can personally look into this matter as I already have in hand the images you kindly provided. https://www.abcam.com/index.html?section=ip&pageconfig=technical&intAbID=19387&mode=questionaire I am already trying to understand what might be happening and would be very grateful if you can clarify a few points: -there are several bands on all blots, I'd be very interrested to know their sizes but with only one MW marker am finding it hard to determine this (are the bands above 78kda close to 100kDa for example, or 80kDa?) -Looking at the western blots of the lysates, I see one band with the Mono (I guess this is the 80Kda band), but two bands both with the poly and the HA tag antibodies. I am wondering if the HA tag is not also a poly. In general polys do pick up better various forms of the proteins and it could be that those two polys are picking up cleaved forms of TEM8. Could this be the case? -Looking at the IPed reprobes, it seems again to me that the poly anti TEM8 and the HA tags are giving similar band patterns, wether it was an IP with HA antibody or the poly. So with the IP with the poly it seems to me that the reprobe with the HA does give something, and as the TEM8 is expressed not endogenously in your cells I am wondering if again it could be because of a cleavage of the protein. Interrestingly IPing with the mono did not give a band at 80kda, so I conclude that the mono does not work well in IP, is this your opinion too? I would be very grateful if you can let me know your thoughts on those points so I can better understand what might be happening in your experiments.

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