CD312 antibody | 2A1
Mouse anti Human CD312 antibody, clone 2A1 (MCA2330) used to evaluate EMR2 expression in treated HEK-293 cell lysates by western blotting.
Image caption:
Glycosylation-dependent membrane-association of EMR2-NTF.
(A) Schematic representation of the chimeric EMR2-WT-PAR1-myc and EMR2-S518A-PAR1-myc proteins. The EGF-like 1, 2 and 5 motifs of EMR2 protein are represented by three triangles. The GPS and thrombin cleavage sites are indicated by a black and a gray arrow, respectively. The myc tag is represented by a black circle. (B) WB analysis of HEK293T cells expressing EMR2-WT-PAR1-myc (#1) and EMR2-S518A-PAR1-myc (#2) treated with or without thrombin. Note the sizes of the thrombin-released NTFs in supernatant (white and black stars) are bigger than the NTFs remained in cell lysate (white and black arrows). (C) Flow cytometry analysis of the surface EMR2 expression of transfected HEK293T cells treated with (dash line) or without (black line) thrombin. Mock transfected HEK293T cells were negative controls. (D) WB analysis of supernatant and cell lysate of thrombin-digested HEK293T cells expressing EMR2-S518A-PAR1-myc treated with indicated de-glycosylation enzymes. The heterodimeric and membrane-associated EMR2-NTF molecules are indicated by a black star and a white star, respectively. (E) WB analysis of supernatant and cell lysate of PFO-treated HEK293T cells. Transfected cells expressing EMR2-WT-myc (#1), EMR2-S518A-myc (#2), EMR2-WT-PAR1-myc (#3) and EMR2-S518A-PAR1-myc (#4) were treated with or without thrombin first, washed and incubated with 0.2% PFO. Supernatant and cell lysate were then collected and analyzed by WB analysis using 2A1 and anti-myc mAbs as indicated. The white arrowhead and asterisk indicate the NTF and CTF of EMR2-WT-myc respectively, whereas the white arrows represent the EMR2-S518A-myc. The black arrow indicates the uncleaved EMR2-S518A-PAR1-myc protein, while the black asterisk indicates the cleaved PAR1 fragment following thrombin digestion.
From: Huang YS, Chiang NY, Chang GW, Lin HH.
Membrane-association of EMR2/ADGRE2-NTF is regulated by site-specific N-glycosylation.
Sci Rep. 2018 Mar 14;8(1):4532.
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Mouse anti Human CD312 antibody, clone 2A1 (MCA2330) used to evaluate EMR2 expression in treated HEK-293 cell lysates by western blotting.
Image caption:
The hydrophilic characteristics of membrane-associated EMR2-NTF. (A) WB analysis of supernatant and cell lysate samples subjected to Triton X-114 phase separation. Supernatant and cell lysate were collected from PFO-treated CHO-K1 cells expressing EMR2-WT-myc. Ctl: input control protein samples before Triton X-114 phase separation; AP: aqueous phase; DP1 and 2: the first and second detergent phases, respectively. (B) WB analysis of membrane fraction of EMR2-WT-myc expressing cells after PBS, high salt (1 M NaCl) and high pH (100 mM Na2CO3, pH 11) treatment. S and L represent supernatant and cell pellets, respectively. The arrow indicates the specific Gαi1,2 protein band. (C) WB analysis of membrane fraction of thrombin-treated (T+) or untreated (T−) EMR2-WT-PAR1-myc expressing cells, followed by PBS, high salt (1 M NaCl) and high pH (100 mM Na2CO3, pH 11) treatment. Blots were probed with indicated mAbs.
From: Huang YS, Chiang NY, Chang GW, Lin HH.
Membrane-association of EMR2/ADGRE2-NTF is regulated by site-specific N-glycosylation.
Sci Rep. 2018 Mar 14;8(1):4532.
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Mouse anti Human CD312 antibody, clone 2A1 (MCA2330) used to evaluate EMR2 expression in treated HEK-293 cell lysates by western blotting and immunofluorescence.
Image caption:
Membrane-association of EMR2-NTF occurs in post-ER compartments. (A) Schematic representation of the chimeric EMR2-CD4 fusion proteins including EMR2-WT-CD4-HA-AATN (1), EMR2-S518A-CD4-HA-AATN (2), EMR2-WT-CD4 -HA-KKTN (3) and EMR2-S518A-CD4-HA-KKTN (4). The CD4 TM region and HA tag are represented by a single gray zig-zag line and a black hexagon, respectively. KKTN and AATN indicate the C-terminal ER retention signal peptide and control peptide, respectively. (B) WB analysis of total cell lysates of CHO-K1 cells expressing the respective chimeric EMR2-CD4 fusion proteins as indicated by the numbers in (A). Lysates were digested without (left panel) or with (right panel) Endo-H. The blot was probed with the 2A1 mAb. (C) FACS analysis of EMR2-NTF expression levels on cell surface and permeabilized whole cell of CKO-K1 cells expressing the respective chimeric EMR2-CD4 fusion proteins as indicated by the numbers in (A). (D) Confocal immunofluorescence analysis of EMR2-NTF expression patterns on cell surface (left panel) and whole cell (right panel) of CKO-K1 cells expressing the respective chimeric EMR2-CD4 fusion proteins as indicated by the numbers in (A). The red color represents EMR2-NTF stained by 2A1 mAb followed by goat anti-mFc IgG-Alexa Flour 647. The green color represents the ER marker expressed by the transfected pCMV-myc-ER-GFP plasmid, while the blue color indicates the nucleus stained by DAPI. Scale: 10 μm.
From: Huang YS, Chiang NY, Chang GW, Lin HH.
Membrane-association of EMR2/ADGRE2-NTF is regulated by site-specific N-glycosylation.
Sci Rep. 2018 Mar 14;8(1):4532.
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Mouse anti Human CD312 antibody, clone 2A1 (MCA2330) used for the evaluation of EMR2 expression in cell lysates by western blotting.
Image caption:
CD4 is shed by MMP-like proteinases in transfected CHO-K1 cells.
(A, B) CHO-K1 cells were transiently transfected with the hCD4-myc or negative control EMR2-myc expression construct. Production of sCD4 is evaluated by ELISA (A) and WB (B) analysis. Conditioned medium (CM) and cell lysates (CL) were probed with 2A1 (specific to EMR2), anti-CD4 and anti-myc mAbs in WB analysis. Kruskal-Wallis test across all three groups of 1X and 10X concentration shows p = 0.027. Dunn’s multiple comparisons post-test p values between groups show ***P<0.001. (C) sCD4 production is inhibited by pan-MMP inhibitors, GM6001 and TAPI-0. CHO-K1 cells transiently transfected with the hCD4-myc construct were treated with various protease inhibitors (1, Control; 2, DMSO; 3, Furin protease inhibitor I; 4, Pepstatin A; 5, GM6001; 6, TAPI-0) then analyzed by WB (left panel) and ELISA (right panel). Kruskal-Wallis test across all six groups shows p = 0.02. Dunn’s multiple comparisons post-test p values between groups show ***P<0.001. (D, E) CHO-K1 cells transiently transfected with the hCD4-myc construct were treated with PMA in various concentrations (D) for 1 hr or 50 nM PMA for different time periods (E) then analyzed by ELISA and WB. ELISA. Statistical significance was assessed by the trend analysis.
From: Tseng W-Y, Huang Y-S, Chiang N-Y, Chou Y-P, Wu Y-JJ, Luo S-F, et al. (2013)
Increased Soluble CD4 in Serum of Rheumatoid Arthritis Patients Is Generated by Matrix Metalloproteinase (MMP)-Like Proteinases.
PLoS ONE 8(5): e63963.
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Mouse anti Human CD312 antibody, clone 2A1 (MCA2330) used to identify EMRs by immunoprecipitation and western blotting.
Image caption:
Detection of EMR2 by commercial antibodies. HEK 293 T cells were transfected with a control BacMam virus (V) or with BacMam virus engineered to produce EMR2 (E). Protein size markers are indicated (M). Immunoprecipitation (IP) was carried out from cell lysates using R&D Systems anti-EMR2 pAb AF4894 (panel a), or Serotec mAb 2A1 (panel b) and immunoprecipitates were subjected to electrophoresis on the same gel. Following transfer, the membrane was divided into two strips which were probed with (a) 2A1, or (b) AF4894, respectively. These strips are presented in their entirety side by side as panels a and b. The specific EMR2 band is boxed in each case.
From: Bhudia N, Desai S, King N, Ancellin N, Grillot D, Barnes AA, Dowell SJ.
G Protein-Coupling of Adhesion GPCRs ADGRE2/EMR2 and ADGRE5/CD97, and Activation of G Protein Signalling by an Anti-EMR2 Antibody.
Sci Rep. 2020 Jan 22;10(1):1004.
doi: 10.1038/s41598-020-57989-6.
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Mouse anti Human CD312 antibody, clone 2A1 (MCA2330) used to activate EMR2 in a functional assay.
Image caption:
Activation of EMR2 by an antibody. NFAT-Luciferase reporter (NFAT-Luc) responses were measured following transient co-transfection of Vector, EMR2-FL, EMR2-CTF or AT-1 constructs with an NFAT-Luciferase reporter construct into HEK 293 T cells, and treatment with EMR2 antibodies (5–10 mg/ml) or Ang II (25 μM) as indicated: (panel b) Serotec mAb, 2A1. Data were normalised as fold effect relative to vector control.
From: Bhudia N, Desai S, King N, Ancellin N, Grillot D, Barnes AA, Dowell SJ.
G Protein-Coupling of Adhesion GPCRs ADGRE2/EMR2 and ADGRE5/CD97, and Activation of G Protein Signalling by an Anti-EMR2 Antibody.
Sci Rep. 2020 Jan 22;10(1):1004.
doi: 10.1038/s41598-020-57989-6.
This image is from an open access article distributed under terms of a Creative Commons Attribution License.
Mouse anti Human CD312
- Product Type
- Monoclonal Antibody
- Clone
- 2A1
- Isotype
- IgG1
- Specificity
- CD312
Antibody Quality and Performance Guarantee
| Mouse anti Human CD312 antibody, clone 2A1 recognizes human EMR2, a member of the epidermal growth factor-seven transmembrane (EGF-TM7) family of proteins, which is closely related to CD97. EMR2, also known as CD312, is predominantly expressed on myeloid dendritic cells, monocytes and tissue macrophages. Various isoforms of EMR2 have been documented. The ligand for the largest isoform of EMR2 has recently been identified as chrondroitin sulphate, which binds to the fourth EGF-like module of EMR2. Mouse anti Human CD312 antibody, clone 2A1 recognizes the stalk region of EMR2. |
- Target Species
- Human
- Product Form
- Purified IgG - liquid
- Preparation
- Purified IgG prepared by affinity chromatography on Protein A from tissue culture supernatant
- Buffer Solution
- Phosphate buffered saline
- Preservative Stabilisers
0.09% Sodium Azide - Carrier Free
- Yes
- Immunogen
- NIH-3T3 cells stably transfected with EMR2 (EGF1-5) cDNA.
- Approx. Protein Concentrations
- IgG concentration 1.0 mg/ml
- Fusion Partners
- Spleen cells from immunized Balb/c mice were fused with cells of the mouse SP2/0 myeloma cell line.
- Regulatory
- For research purposes only
- Guarantee
- 12 months from date of despatch
This product is shipped at ambient temperature. It is recommended to aliquot and store at -20°C on receipt. When thawed, aliquot the sample as needed. Keep aliquots at 2-8°C for short term use (up to 4 weeks) and store the remaining aliquots at -20°C.
Avoid repeated freezing and thawing as this may denature the antibody. Storage in frost-free freezers is not recommended.
Avoid repeated freezing and thawing as this may denature the antibody. Storage in frost-free freezers is not recommended.
This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
| Application Name | Verified | Min Dilution | Max Dilution |
|---|---|---|---|
| Flow Cytometry |  |
1/50 | 1/200 |
| Immunohistology - Frozen | |
1/50 | 1/200 |
| Immunoprecipitation | |
||
| Western Blotting | |
Where this antibody has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. It is recommended that the user titrates the antibody for use in their own system using appropriate negative/positive controls.
- Flow Cytometry
- Use 10ul of the suggested working dilution to label 106 cells in 100ul.
| Description | Product Code | Applications | Pack Size | List Price | Your Price | Quantity | |
|---|---|---|---|---|---|---|---|
| Mouse IgG1 Negative Control | MCA928 | F | 100 Tests |
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| Description | Mouse IgG1 Negative Control | ||||||
References for CD312 antibody
-
Kwakkenbos, M.J. et al. (2002) The human EGF-TM7 family member EMR2 is a heterodimeric receptor expressed on myeloid cells.
J Leukoc Biol. 71 (5): 854-62. -
Stacey, M. et al. (2003) The epidermal growth factor-like domains of the human EMR2 receptor mediate cell attachment through chondroitin sulfate glycosaminoglycans.
Blood. 102 (8): 2916-24. -
Lin, H.H. et al. (2004) Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif.
J Biol Chem. 279 (30): 31823-32. -
Yona, S. et al. (2008) Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function.
FASEB J. 22 (3): 741-51. -
Tseng, W.Y. et al. (2013) Increased soluble CD4 in serum of rheumatoid arthritis patients is generated by matrix metalloproteinase (MMP)-like proteinases.
PLoS One. 8 (5): e63963. -
Huang, Y.S. et al. (2018) Membrane-association of EMR2/ADGRE2-NTF is regulated by site-specific N-glycosylation.
Sci Rep. 8 (1): 4532. -
Shankar-Hari, M. et al. (2018) Early PREdiction of sepsis using leukocyte surface biomarkers: the ExPRES-sepsis cohort study.
Intensive Care Med. 44 (11): 1836-48. -
Bhudia, N. et al. (2020) G Protein-Coupling of Adhesion GPCRs ADGRE2/EMR2 and ADGRE5/CD97, and Activation of G Protein Signalling by an Anti-EMR2 Antibody.
Sci Rep. 10 (1): 1004.
View The Latest Product References
Further Reading
-
Kwakkenbos, M.J. et al. (2004) The EGF-TM7 family: a postgenomic view.
Immunogenetics. 55 (10): 655-66.
- Synonyms
- EMR2
- RRID
- AB_566724
- UniProt
- Q9UHX3
- Entrez Gene
- EMR2
- GO Terms
- GO:0005886 plasma membrane
- GO:0005509 calcium ion binding
- GO:0016021 integral to membrane
- GO:0004930 G-protein coupled receptor activity
- GO:0007218 neuropeptide signaling pathway
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