Overview

  • Product name
    Anti-M6PR (cation independent) antibody [2G11]
    See all M6PR (cation independent) primary antibodies
  • Description
    Mouse monoclonal [2G11] to M6PR (cation independent)
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, IHC-Fr, IPmore details
    Unsuitable for: WB
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, African green monkey
    Predicted to work with: Non human primatesDoes not react with: Hamster
  • Immunogen

    Purified bovine 300 kDa Mannose 6 Phosphate Receptor (Cation independent).

  • Epitope
    This antibody is shown to recognize an epitope in the extracellular domain of Mannose 6 Phosphate Receptor.
  • Positive control
    • In Flow Cytometry, this antibody gave a positive signal in A431 cells.
  • General notes

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab2733 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
Flow Cyt Use 1µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

ICC/IF Use a concentration of 5 - 10 µg/ml.
IHC-Fr Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
  • Application notes
    Is unsuitable for WB.
  • Target

    • Function
      Transport of phosphorylated lysosomal enzymes from the Golgi complex and the cell surface to lysosomes. Lysosomal enzymes bearing phosphomannosyl residues bind specifically to mannose-6-phosphate receptors in the Golgi apparatus and the resulting receptor-ligand complex is transported to an acidic prelyosomal compartment where the low pH mediates the dissociation of the complex. This receptor also binds IGF2. Acts as a positive regulator of T-cell coactivation, by binding DPP4.
    • Sequence similarities
      Belongs to the MRL1/IGF2R family.
      Contains 1 fibronectin type-II domain.
    • Domain
      Contains 15 repeating units of approximately 147 AA harboring four disulfide bonds each. The most highly conserved region within the repeat consists of a stretch of 13 AA that contains cysteines at both ends.
    • Cellular localization
      Lysosome membrane. Colocalized with DPP4 in internalized cytoplasmic vesicles adjacent to the cell surface.
    • Information by UniProt
    • Database links
    • Alternative names
      • 300 kDa mannose 6 phosphate receptor antibody
      • 300 kDa mannose 6-phosphate receptor antibody
      • Cation independent mannose 6 phosphate receptor antibody
      • Cation-independent mannose-6-phosphate receptor antibody
      • CD222 antibody
      • CD222 antigen antibody
      • CI Man 6 P receptor antibody
      • CI Man-6-P receptor antibody
      • CI MPR antibody
      • CI-M6PR antibody
      • CI-MPR antibody
      • CIMPR antibody
      • IGF 2 receptor antibody
      • IGF 2R antibody
      • IGF II receptor antibody
      • IGF-II receptor antibody
      • IGF2 receptor antibody
      • Igf2r antibody
      • Insulin like growth factor 2 receptor antibody
      • Insulin like growth factor II receptor antibody
      • Insulin-like growth factor 2 receptor antibody
      • Insulin-like growth factor II receptor antibody
      • M6P R antibody
      • M6P/IGF2 receptor antibody
      • M6P/IGF2R antibody
      • M6PR antibody
      • mannose 6 phosphate receptor antibody
      • mannose 6 phosphate receptor, cation independent antibody
      • MPR 300 antibody
      • MPR300 antibody
      • MPRI antibody
      • MPRI_HUMAN antibody
      see all

    Images

    • ICC/IF image of ab2733 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab2733, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

    • Overlay histogram showing HeLa cells stained with ab2733 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2733, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
    • Immunofluorescent imaging of human cells (U2OS) with ab2733 confirms the specificity of this antibody, with the expected perinuclear vesicular staining of lysosomes. 

      IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes.  All blocking and incubation steps carried out at 37 degrees.

    • Immunostained frozen sections showing that mannose 6 phosphate receptor is localised in the outermost epithelial cell layer.

      This picture was kindly supplied as part of the review submitted by Marko Nykanen.

    • ab2733 positively staining formaldehyde fixed Human HEK 293 cells (red) in conjunction with goat anti mouse (Alexa 546). Nuclear staining was obtained using Hoechst.
      This image is an edited version of an image received courtesy of an Abreview submitted by Kun Liu on 19 September 2005. We do not have any further information relating to this image.

      See Abreview

    • ICC/IF image of ab2733 stained HepG2 cells. The cells were 4% PFA 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 (ab2733, 10µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) 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:
    • Navarro Negredo P  et al. The WDR11 complex facilitates the tethering of AP-1-derived vesicles. Nat Commun 9:596 (2018). Read more (PubMed: 29426865) »
    • Newton T  et al. Mechanistic basis of an epistatic interaction reducing age at onset in hereditary spastic paraplegia. Brain 141:1286-1299 (2018). Read more (PubMed: 29481671) »
    See all 70 Publications for this product

    Customer reviews and Q&As

    1-10 of 32 Abreviews or Q&A

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Permeabilization
    Yes - 0.1% Triton X-100
    Specification
    HeLa
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Oct 11 2018

    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 xxx with ab124767.

    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.

    Read More

    Answer

    Thank you, I received the data this time. For a protocol modification, I suggest trying preparing a sample by heating at 60C for 10 minutes instead of 95 for 5 minutes to minimize aggregation of the trans-membrane domains. It is strange that both antibodies give you this result, and I still think there may be some issue with the sample entering the gel and/or transferring effectively, even though the standards do.

    Another modification is to remove methanol from the transfer buffer and add SDS to a final concentration of 0.1%, to encourage transfer of this large protein.

    Are you confident that the secondary antibody is not reacting with the samples? Is the secondary specific for a subclass of mouse IgG?

    These modifications are unlikley to remove the band at 70 kDa. We do have other antibodies against the receptor, so if these modifications fail, I can send a vial of something else to try, such as ab32815 or ab124767.

    Read More

    Answer

    Thank you for bringing this to our attention. I will need a few more details of your protocol. I was not able to open the file you sent.

    Can you please tell me how you prepared your lysates (did you use protease inhibitors), and how you heated the samples to denature. I am concerned that the receptor may have aggregated upon boiling and did not enter the gel effectively, or did not transfer to the blot well. Did you check for efficacy of transfer with a Ponceau Red stain or equivalent?

    How much sample did you load per lane of the gel? How did you block the membrane? What concentration of antibody did you apply to the membrane and how long did you incubate with the antibody?

    Read More

    Question
    Answer

    Thank you for contacting us regarding ab2733. I have confirmed with the laboratory that this particular antibody is only batch tested in IF, but has been tested in WB. I have attached an image for you. We have recently done some optimisation with TA gels (and 0.45 um membranes) and we found using 10% methanol +0.04% SDS or 10% ethanol +0.04% SDS for the transfer buffer worked really well. We also found that it was best not to heat the lysates to 100C for TA gels (no heating was best but 60C was ok). We haven’t tried this procedure with this ab though so I can’t say if it will help solve your problem for sure. The presence of SDS increases solubility of membrane proteins and high MW proteins allowing efficient elution from the gel. As standard we denature at 95C. Reduced denaturation temperature (60-70C) or no denaturation is often recommended for membrane proteins/high MW proteins to reduce precipitation and failure of the protein to migrate onto the gel during electrophoresis. I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions or if your results do not improve with this antibody.

    Read More

    Answer

    I'm glad that you have been seeing better results with blocking with BSA. If you are now having difficulty with non-specific bands, increasing the dilution of the primary and secondary antibody as well as adding 1 % BSA and 0.2% Tween 20 into the diluents may help. If you would like me to have a look at your protocol to see if there is any other advice I can think of then please do fill in the form and I'd be happy to try and help. Otherwise, I wish you all the best with your experiments.

    Read More

    Answer

    Thank you for your reply. 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 for ab8093. 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.  

    Read More

    Answer

    Thank you for your reply. As this antibody, ab2733 is a monoclonal antibody I would not expect much lot to lot variation.  It is possible that there was something wrong with just this particular vial as we have not received reports of other problems with this antibody.  If you would prefer to try a different antibody, I can recommend another mouse monoclonal antibody, Anti-Mannose 6 Phosphate Receptor (Cation independent) antibody [MEM-238] (ab8093).  This antibody has been validated for ICC/IF and should also perform well. Please let me know which you would prefer.  I look forward to your reply so that I may assist you further.

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Cow Cell (MDBK - Madin-Darby Bovine Kidney)
    Specification
    MDBK - Madin-Darby Bovine Kidney
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.5% Triton X-100
    Blocking step
    Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Jun 13 2011

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HEK293)
    Specification
    HEK293
    Fixative
    100% EtOH
    Permeabilization
    Yes - 0.1% Triton X-100
    Blocking step
    Gelatin as blocking agent for 40 minute(s) · Concentration: 0.1% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Jan 14 2011

    1-10 of 32 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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