Anti-M6PR (cation independent) antibody [2G11] (ab2733)

Abreviews (12)Q&A (19)Specific References (68)

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, WB, IHC-Fr, IPmore details
  • 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
    • This antibody gave a positive signal in the following whole cell lysates: HeLa, SHSY-5Y, HepG2, MEF1. 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.
WB Use a concentration of 5 - 10 µg/ml. Detects a band of approximately 300 kDa (predicted molecular weight: 300 kDa). Abcam recommends using BSA as the blocking agent.
IHC-Fr Use at an assay dependent concentration.
IP Use at an assay dependent concentration.

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
    see all
  • 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

  • Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [2G11] (ab2733)

    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).

  • Flow Cytometry - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    Flow Cytometry - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    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.
  • Western blot - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    Western blot - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    Lanes 1-4 : Anti-M6PR (cation independent) antibody [2G11] (ab2733) at 5 µg/ml
    Lanes 5-8 : Anti-M6PR (cation independent) antibody [2G11] (ab2733) at 10 µg/ml

    Lanes 1 & 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lanes 2 & 6 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
    Lanes 3 & 7 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lanes 4 & 8 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

    Lysates/proteins at 40 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 300 kDa
    Observed band size: 300 kDa


    Exposure time: 1 minute
  • Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [2G11] (ab2733)Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK

    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.

  • Immunohistochemistry (Frozen sections) - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    Immunohistochemistry (Frozen sections) - Anti-M6PR (cation independent) antibody [2G11] (ab2733)

    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [2G11] (ab2733)

    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

  • Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [2G11] (ab2733)
    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 68 Publications for this product

Publishing research using ab2733? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

Submit a review Submit a question

31 reviews or Q&A

10
Votes: Highest First

ICC/IF with rat neuron priamry culture
no signal at all
fix: 2% PFA 20 min
Ab: 1/50, 1/100, 1/400, 1/800 1h RT and o/n 4 degree
perm: 0.2% TX100 15 min
2nd: works well for other Abs, 488

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 h...

Read More

Brief protocol:



1. 50,000 - 150,000 Cells lysed in 20uL running buffer containing mercaptoethanol for 5min in 95C heat block (no protease inhibitor used)



2. Samples (20uL) run on gel with protein ladder for...

Read More

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 antib...

Read More

I have been having trouble with two Abcam IGF2R (CI-M6PR) antibodies, a b2733 and ab8093, which I have been attempting to use for Western Blotting.

Both antibodies have failed to show a band in the expected region (approx 250Kd) but have ins...

Read More

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 heat...

Read More

I am using this antibody in WB, but am unable to detect any bands.

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 ...

Read More

Thanks, I have just tried with BSA as blocking agent and I did have more success but with also more a specific bands from ms9-II cells that overexpress M6PR. I will also try ab32815 to see if the later ab shows less a specific bands with my ms9-II cell...

Read More

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 dilue...

Read More

Thanks for your advice. If possible I would prefer to try the alternative antibody you suggest (ab8093). Would you be able to send me a replacement vial?

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...

Read More

Many thanks for offering to replace the antibody. Would you recommend that I try a different batch of the same antibody or try a different antibody altogether?

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 problem...

Read More

Immunocytochemistry/ Immunofluorescence abreview for Anti-Mannose 6 Phosphate Receptor (Cation independent) antibody [2G11] - Late Endosome Marker

 Excellent
Abreviews
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
Read More
abreview image
Username

Abcam user community

Verified customer

Submitted Jun 13 2011

Immunocytochemistry/ Immunofluorescence abreview for Anti-Mannose 6 Phosphate Receptor (Cation independent) antibody [2G11] - Late Endosome Marker

 Good
Abreviews
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
Read More
abreview image
Username

Abcam user community

Verified customer

Submitted Jan 14 2011

Immunocytochemistry/ Immunofluorescence abreview for Anti-Mannose 6 Phosphate Receptor (Cation independent) antibody [2G11] - Late Endosome Marker

 Excellent
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa cells)
Specification
HeLa cells
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
Serum as blocking agent for 10 minute(s) · Concentration: 10% · Temperature: 20°C
Read More
abreview image
Username

Dr. Margaret Robinson

Verified customer

Submitted Dec 02 2010

1-10 of 31 Abreviews or Q&A

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

Sign up