Overview

  • Product name

    Anti-M6PR (cation independent) antibody
    See all M6PR (cation independent) primary antibodies
  • Description

    Rabbit polyclonal to M6PR (cation independent)
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ICC, IP, ICC/IF, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Cow, Human, Pig, Non human primates
  • Immunogen

    Full length native protein purified from adult Bovine liver tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab32815 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
WB 1/2000 - 1/3000. Detects a band of approximately 300 kDa (predicted molecular weight: 275 kDa).
ICC 1/50 - 1/500.
IP 1/1000 - 1/2000.
ICC/IF 1/50 - 1/500.
IHC-P 1/1000.

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

  • Immunofluorescent analysis of Mannose 6 Phosphate Receptor (Cation independent) (green) showing staining in the cytoplasm of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Mannose 6 Phosphate Receptor (Cation independent) antibody (ab32815) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

  • Immunofluorescent analysis of Mannose 6 Phosphate Receptor (Cation independent)(green) showing staining in the cytoplasm and nucleus of MCF-7 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Mannose 6 Phosphate Receptor (Cation independent) antibody (ab32815) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

  • Immunofluorescent analysis of Mannose 6 Phosphate Receptor (Cation independent)  (green) showing staining in the cytoplasm of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Mannose 6 Phosphate Receptor (Cation independent) antibody (ab32815) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

  • Ab32815 staining human normal left ventricle of heart. Staining is localized to lysosome and lysosomal membrane.
    Left panel: with primary antibody duluted at 1:1000. Right panel: isotype control.
    Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplifi
  • ICC/IF image of ab32815 stained HeLa 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 (ab32815, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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 19 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (293T)
Gel Running Conditions
Reduced Denaturing
Loading amount
40 µg
Specification
293T
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 08 2019

Application
Western blot
Sample
Sheep Tissue lysate - whole (Heart)
Gel Running Conditions
Non-reduced Denaturing (10%)
Loading amount
75 µg
Specification
Heart
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Abcam user community

Verified customer

Submitted Aug 09 2017

Answer

Thank you for your reply.

If you would prefer, I would be happy to issue ab32815 as a free of charge replacement for ab129923.

I look forward to your reply. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. The one protocol modification that might have an effect on the background stain would be to substitute either a different serum or BSA (1%) for the blocking donkey serum, but if you have not seen this issue with other antibodies in the same protocol with the same reagents, I am doubtful the susbstitution will help.

I have issued a free of charge replacement with the order number 1144831.

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.

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Application
Immunocytochemistry/ Immunofluorescence
Sample
Pig Cell (primary alveolar macrophage)
Specification
primary alveolar macrophage
Fixative
Paraformaldehyde
Permeabilization
Yes - TX-100
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Feb 05 2009

Application
Western blot
Sample
Human Cell lysate - other (Membrane fraction of Hela Cells)
Loading amount
100000 cells
Specification
Membrane fraction of Hela Cells
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Sep 12 2008

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Hela Cells)
Specification
Hela Cells
Fixative
Methanol
Permeabilization
No

Abcam user community

Verified customer

Submitted Aug 11 2008

Application
Western blot
Sample
Mouse Cell lysate - whole cell (lacrimal gland)
Loading amount
25 µg
Specification
lacrimal gland
Gel Running Conditions
Reduced Denaturing
Blocking step
Rockland Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Jul 10 2008

Answer

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab32815 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. What species of cell extract are you using? In most cases, the cause of multiple bands is because the primary and/or secondary antibody concentration is too high and causes non-specific binding. The dilution that we have on the datasheet is a recommended starting dilution and customers are encouraged to determine the optimal concentration/dilution. You can try decreasing the primary (1:2000 ~ 1:5000). Also, running a no-primary control experiment will be able to eliminate the possibility that your secondary antibody is binding non-specifically. What are the band sizes you obtained? The bands located above the expected molecular weight band could be multimers. Please try boiling your protein in SDS-PAGE for 10 minutes to ensure proper disruption of multimers. This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. Meanwhile, bands located below the expected molecular weight may indicate that your target protein has been digested. Please make sure that you incorporated sufficient protease inhibitors and have kept your samples on ice the whole time. If you can provide an image, that would certainly help me understand the problem better. Since you mentioned that you seeing high background, I would assume that it is the milk that is causing the problem. In general, there are two blocking solutions that are widely used, non-fat milk and BSA. We would recommend using 5% BSA as almost all of our products are tested with it and have proven to be effective. Most customers who use milk usually have high background, due to the secondary antibody binding to the milk. You can check if this dark signal you are detecting is from the secondary or not by incubating your blot in secondary only. Increasing the washing frequency and duration would also help eliminate high background. Can you confirm that you have used Tween in your washing solution and have washed at least 15min x 3 times? Please try this if you have not already done so. I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with the answers to the above and your shipping address. Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.

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