Overview

  • Product name
    Anti-M6PR (cation independent) antibody [MEM-238]
    See all M6PR (cation independent) primary antibodies
  • Description
    Mouse monoclonal [MEM-238] to M6PR (cation independent)
  • Host species
    Mouse
  • Specificity
    CD222 antigen (human)
  • Tested applications
    Suitable for: Flow Cyt, IP, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Non human primates
  • Immunogen

    Vaccinia-M6PR/iGFIIR

  • Epitope
    Recognizes an epitope between domains 2 and 5.
  • General notes

     

    CD222 is a 250kDa transmembrane protein with a short cytoplasmic tail containing an internalization signal. CD222 was originally identified as a receptor for IGFII and M6P-containing proteins (e.g. lysosomal hydrolases). Lysosomal enzymes are sorted to lysosomes via CD222 either from the Golgi, where the enzymes acquire M6P, or from the extracellular space. The majority of CD222 molecules (approximately 90-95%) are located intracellularly, only 5-10% is present on the cell membrane. The internalization rate seems to be enhanced by ligand induced dimerization of CD222 as well as by phosphorylation of its cytoplasmic serine. CD222 is also a receptor for TGFbeta latency associated peptide (LAP), proliferin and may bind several molecules independently of M6P, including plasminogen, CD87 or retinoic acid. It is involved in activation of latent TGFbeta [PROW].

    This product was changed from ascites to tissue culture supernatant on 24th January 2018. Please note that the dilutions may need to be adjusted accordingly. If you have any questions, please do not hesitate to contact our scientific support team.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.40
    Preservative: 0.097% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    >95% by SDS-PAGE
  • Purification notes
    Purified from TCS
  • Primary antibody notes
    CD222 is a 250kDa transmembrane protein with a short cytoplasmic tail containing an internalization signal. CD222 was originally identified as a receptor for IGFII and M6P-containing proteins (e.g. lysosomal hydrolases). Lysosomal enzymes are sorted to lysosomes via CD222 either from the Golgi, where the enzymes acquire M6P, or from the extracellular space. The majority of CD222 molecules (approximately 90-95%) are located intracellularly, only 5-10% is present on the cell membrane. The internalization rate seems to be enhanced by ligand induced dimerization of CD222 as well as by phosphorylation of its cytoplasmic serine. CD222 is also a receptor for TGFbeta latency associated peptide (LAP), proliferin and may bind several molecules independently of M6P, including plasminogen, CD87 or retinoic acid. It is involved in activation of latent TGFbeta [PROW].
  • Clonality
    Monoclonal
  • Clone number
    MEM-238
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab8093 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.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

IP Use a concentration of 10 µg/ml.
WB Use at an assay dependent concentration. Use under non reducing condition.
ICC/IF Use at an assay dependent concentration. Used at a concentration of 5 ug/ml for 1 hr on HEK cells (see Abreview for further information).

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

  • ab8093 at 5µg/ml staining human HEK cells by immunocytochemistry. The cells were fixed with paraformaldehyde and incubated with the antibody for 1 hour. Bound antibody was detected using a goat anti-mouse IgG Alexa-Fluor ® 568. In the confocal image ab8093 labelling in red shows a distribution consistent with the location of the trans-Golgi network and lysosomes. Blue nuclear counterstain is present.

    This image is courtesy of an Abreview submitted by Randal Moldrich on 31 March 2006.

    See Abreview

  • ab8093 staining Mannose 6 Phosphate Receptor (Cation independent) in human HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 and blocked with 2% BSA for 30 minutes at 25°C . Samples were incubated with primary antibody (1/300 in 2%BSA + 0.1% Triton X-100) for 2 hours at 25°C. An Alexa Fluor® 488-conjugated Goat polyclonal to mouse IgG (dilution 1/2000) was used as secondary antibody.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab8093 (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 (ab8093, 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 IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References

This product has been referenced in:
  • Hirst J  et al. Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval. PLoS Biol 16:e2004411 (2018). WB ; Human . Read more (PubMed: 29381698) »
  • Stadlmann J  et al. Comparative glycoproteomics of stem cells identifies new players in ricin toxicity. Nature 549:538-542 (2017). Read more (PubMed: 28959962) »
See all 12 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer

I have issued a free of charge replacement with the order number for ab32815.

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

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

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 contacting us. Having looked into the case a little I can see no reason why ab8093 should not be performing well with Western blotting or that ab2733 would be likely to perform any better. What might be helpful is if you were to share the protocol which you are currently performing and I may be able to spot some things which may help to improve the performance of ab8093. To this end I have attached a questionnaire, this should only take 5-10 minutes to fill in. It would be helpful if you could also attach an image of the blot obtained. This information will also help us to monitor the quality of our products and to determine if any further internal testing needs to be performed. If the antibody is not performing to the level we would expect with the applications and species listed on the datasheet you would be eligible for a replacement antibody or refund. We do have an antibody in our catalogue which have been more fully characterised with Western blotting and may serve as an alternative to ab8093 if necessary. Ab32815 has been used by one of our customers to detect the Mannose 6 Phosphate Receptor protein from the membrane fraction of Hela Cells using Western blotting: https://www.abcam.com/index.html?datasheet=32815&tab=abreviews&intabreviewid=11713 I'm sorry that I could not be of more help but hopefully with the protocol in hand we can decide if trying an alternative antibody would be worthwhile. In the meantime I am sorry for any inconvenience this has caused.

Read More

Question
Answer

I have attached an Excel file with information regarding all our current antibodies that have been tested in EM. I hope this is helpful. Please let me know if you have any further questions.

Read More

Answer

Thank you for your enquiry. I am working up an internal query to obtain a list of all EM-tested antibodies in our catalog. I will forward you this list as soon as possible. Thanks for your patience!

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (A549)
Specification
A549
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton
Blocking step
5% Goat Serum, 0.5% BSA, 0.5% Gelatin as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Jul 07 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton X-100
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 11 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK cells)
Specification
HEK cells
Fixative
Paraformaldehyde
Blocking step
BSA as blocking agent for 40 minute(s) · Concentration: 5%

Dr. Randal Moldrich

Verified customer

Submitted Mar 31 2006

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

Sign up