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  1. Link

    m6pr-cation-independent-antibody-2g11-ab2733.pdf

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Immunology Innate Immunity Macrophage / Inflamm.
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Validated using a knockout cell line

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

  • Datasheet
  • SDS
Reviews (18)Q&A (19)References (115)

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

Key features and details

  • Mouse monoclonal [2G11] to M6PR (cation independent)
  • Suitable for: Flow Cyt, ICC/IF
  • Knockout validated
  • Reacts with: Human
  • Isotype: IgG2a

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 647

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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/IFmore details
    Unsuitable for: WB
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat, Cow, Non human primates, African green monkeyDoes not react with: Hamster
  • Immunogen

    Full length protein. This information is proprietary to Abcam and/or its suppliers.

  • 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. ICC/IF: HAP1 cells (HAP1-IGF2R knockout cells used as negative cell line)
  • General notes

    This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

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: 0.02% Sodium azide
    Constituents: PBS, 6.97% L-Arginine
  • Concentration information loading...
  • Purity

    Protein G purified
  • Clonality

    Monoclonal
  • Clone number

    2G11
  • Isotype

    IgG2a
  • Research areas

    • Immunology
    • Innate Immunity
    • Macrophage / Inflamm.
    • Tags & Cell Markers
    • Subcellular Markers
    • Organelles
    • Endosome
    • Signal Transduction
    • Protein Trafficking
    • Golgi Proteins
    • Neuroscience
    • Processes

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-M6PR (cation independent) antibody [2G11] (ab205812)
    • Alexa Fluor® 647 Anti-M6PR (cation independent) antibody [2G11] (ab205813)
  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
    • Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879)
  • Isotype control

    • Mouse IgG2a, kappa monoclonal [MG2a-53] - Isotype control (ab18415)

Applications

The Abpromise guarantee

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 (15)
Use a concentration of 1 - 10 µg/ml.
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 1 - 10 µg/ml.
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.
  • Target information above from: UniProt accession P11717 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 281849 Cow
    • Entrez Gene: 3482 Human
    • Entrez Gene: 16004 Mouse
    • Entrez Gene: 25151 Rat
    • Omim: 147280 Human
    • SwissProt: P08169 Cow
    • SwissProt: P11717 Human
    • SwissProt: Q07113 Mouse
    • Unigene: 487062 Human
    • Unigene: 673278 Human
    • Unigene: 26553 Mouse
    • Unigene: 486511 Mouse
    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)

    ab2733 staining IGF2R in wild-type HAP1 cells (top panel) and IGF2R knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab2733 at 1ug/ml and ab6046 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • 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.
  • 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.

  • 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.

Protocols

  • Mouse on Mouse staining protocol

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (115)

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

ab2733 has been referenced in 115 publications.

  • Liu Y  et al. Membrane skeleton modulates erythroid proteome remodeling and organelle clearance. Blood 137:398-409 (2021). PubMed: 33036023
  • Wang H  et al. Unraveling GLUT-mediated transcytosis pathway of glycosylated nanodisks. Asian J Pharm Sci 16:120-128 (2021). PubMed: 33613735
  • Bieri J  et al. Human parvovirus B19 interacts with globoside under acidic conditions as an essential step in endocytic trafficking. PLoS Pathog 17:e1009434 (2021). PubMed: 33878123
  • Hess MW  et al. Advanced Microscopy for Liver and Gut Ultrastructural Pathology in Patients with MVID and PFIC Caused by MYO5B Mutations. J Clin Med 10:N/A (2021). PubMed: 33924896
  • Naik S  et al. A 3D Renal Proximal Tubule on Chip Model Phenocopies Lowe Syndrome and Dent II Disease Tubulopathy. Int J Mol Sci 22:N/A (2021). PubMed: 34069732
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 37 Abreviews or Q&A

Immunocytochemistry/ Immunofluorescence abreview for Anti-M6PR (cation independent) antibody [2G11]

Excellent
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK293T)
Permeabilization
Yes - 0.1% Triton in PBS (5 mins)
Specification
HEK293T
Blocking step
BSA as blocking agent for 15 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Dec 18 2020

Western blot abreview for Anti-M6PR (cation independent) antibody [2G11]

Good
Abreviews
Abreviews
This product is known to not work in this application or species.
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
HEK293
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Abcam user community

Verified customer

Submitted Dec 18 2020

Immunocytochemistry/ Immunofluorescence abreview for Anti-M6PR (cation independent) antibody [2G11]

Excellent
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (RPE-1 cell)
Permeabilization
Yes - 0.3% tritonx100
Specification
RPE-1 cell
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Nov 27 2019

Immunocytochemistry/ Immunofluorescence abreview for Anti-M6PR (cation independent) antibody [2G11]

Good
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (MEFs)
Permeabilization
Yes - 0.1%Triton
Specification
MEFs
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Jul 18 2019

Immunocytochemistry/ Immunofluorescence abreview for Anti-M6PR (cation independent) antibody [2G11]

Excellent
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Permeabilization
Yes - TritonX100
Specification
HeLa
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 22°C
Fixative
Paraformaldehyde
Read More

DR. Jerome Cattin

Verified customer

Submitted Jun 19 2019

Immunocytochemistry/ Immunofluorescence abreview for Anti-M6PR (cation independent) antibody [2G11]

Excellent
Abreviews
Abreviews
abreview image
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
Read More

Abcam user community

Verified customer

Submitted Oct 11 2018

Question

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

Read More

Abcam community

Verified customer

Asked on Nov 28 2012

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

Abcam Scientific Support

Answered on Nov 28 2012

Question

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 approx 90 mins.



3. Transferred for 2hr - very good transfer of protein marker seen



4. Blocked for 60 mins at RT with 5% milk



5. Incubated with primary antibody (1 in 2000 dilution) overnight at 4C



6. Incubated with secondary antibody for 1hr at RT



7. ECL applied for 1 min



8. Membrane developed (images below).

Read More

Abcam community

Verified customer

Asked on Mar 02 2012

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

Abcam Scientific Support

Answered on Mar 02 2012

Question

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 instead shown a band at around 70Kd. I have confirmed the cell lines that I am using (CMK and K562) express IGF2R using PCR and flow cytometry. I have attached a document containing the gels run for the two proteins. I have tried each antibody 3 times now with similar results. I have run secondary only controls, which do not show this band.

Do you have any suggestions on how to improve my results?

Read More

Abcam community

Verified customer

Asked on Mar 02 2012

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

Abcam Scientific Support

Answered on Mar 02 2012

Question

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

Read More

Abcam community

Verified customer

Asked on Dec 21 2011

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

Abcam Scientific Support

Answered on Dec 21 2011

1-10 of 37 Abreviews or Q&A

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