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92114
Human TREM2 Activity Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Human TREM2 Activity Antibody Sampler Kit #92114

Citations (0)
Confocal immunofluorescent analysis of SW620 cells (left, positive) and ACHN cells (right, negative) using Syk (D3Z1E) XP® Rabbit mAb (green), DyLight 650 Phalloidin #12956 (red), and DAPI #4083 (blue).
Simple Western™ analysis of lysates (0.1 mg/mL) from Raji cells using Syk (D3Z1E) XP® Rabbit mAb #13198. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western blot analysis of extracts from various cell lines using Syk (D3Z1E) XP® Rabbit mAb.
Western blot analysis of extracts from Ramos cells, untreated or treated with anti-IgM, using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (upper) or Syk Antibody #2712 (lower).
Western blot analysis of extracts from THP-1 and SH-SY5Y cells, untreated (-) or treated with peptide N-glycosidase F (PNGase F; +), using TREM2 (E9U8L) Rabbit mAb (Amino-terminal Antigen) (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from various cell lines using CD33 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human TREM2 protein (hTREM2-Myc; +), using TREM2 (D8I4C) Rabbit mAb.
Immunoprecipitation of DAP12 protein from THP-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is DAP12 (E7U7T) Rabbit mAb. Western blot analysis was performed using DAP12 (D7G1X) Rabbit mAb #12492. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Western blot analysis of extracts from various cell lines using DAP12 (E7U7T) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (left, positive) and SNB-19 cells (right, negative) using Dap12 (E7U7T) Rabbit mAb (green) and DAPI #4083 (blue).
Immunoprecipitation of Syk protein from SR cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Syk(D3Z1E) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Syk (D3Z1E) XP® Rabbit mAb
Western blot analysis of extracts from THP-1 WT (left) or SYK KO (right) using Syk (D3Z1E) XP® Rabbit mAb (upper). Membranes stained with Ponceau S for total protein normalization (lower).  These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies, as a companion to validation data generated by CST scientists.
Confocal immunofluorescent analysis of Ramos cells, serum-starved (overnight; left) or IgM-treated (12 ug/ml, 2 minutes; right), using Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunoprecipitation of CD33 protein from TF-1 cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is CD33 Antibody. Western blot analysis was performed using CD33 Antibody. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Western blot analysis of extracts from THP-1, HL-60, and Jurkat cells using TREM2 (D8I4C) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Syk (D3Z1E) XP® Rabbit mAb.
Flow cytometric analysis of Ramos cells, untreated (blue) or treated with anti-IgM (green), using Phospho-Syk(Tyr525/526) (C87C1) Rabbit mAb, or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8885 was used as a secondary antibody.
Immunoprecipitation of TREM2 from THP-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TREM2 (D8I4C) Rabbit mAb. Western blot analysis was performed using TREM2 (D8I4C) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lymph node using Syk (D3Z1E) XP® Rabbit mAb.
Confocal immunofluorescent analysis of THP-1 (positive, left) and HL-60 (negative, right) cells using TREM2 (D8I4C) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded mouse spleen using Syk (D3Z1E) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Flow cytometric analysis of RL cells using Syk (D3Z1E) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.
To Purchase # 92114
Cat. # Size Qty. Price Inventory
92114T
1 Kit  (6 x 20 microliters)

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
TREM2 (D8I4C) Rabbit mAb 91068 20 µl
  • WB
  • IP
  • IF
H 28 Rabbit IgG
TREM2 (E9U8L) Rabbit mAb (Amino-terminal Antigen) 70551 20 µl
  • WB
H 28 Rabbit IgG
CD33 Antibody 77576 20 µl
  • WB
  • IP
H 70-80 Rabbit 
Syk (D3Z1E) XP® Rabbit mAb 13198 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R 72 Rabbit IgG
Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb 2710 20 µl
  • WB
  • IP
  • IF
  • F
H 72 Rabbit IgG
DAP12 (E7U7T) Rabbit mAb 97415 20 µl
  • WB
  • IP
  • IF
H 10, 12 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Rab Goat 

Product Description

The Human TREM2 Activity Antibody Sampler Kit provides an economical means of evaluating key members of the human TREM2 signaling pathway using phospho-specific and control antibodies. The kit includes enough antibodies to perform two western blot experiments with each primary antibody. 


Specificity / Sensitivity

Each antibody in the Human TREM2 Activity Antibody Sampler Kit detects endogenous levels of its target protein. TREM2 (D8I4C) Rabbit mAb recognizes endogenous levels of total TREM2 protein. TREM2 (E9U8L) Rabbit mAb (Amino-terminal Antigen) recognizes endogenous levels of total TREM2 protein. This protein does not cross-react with mouse TREM2. A non-specific band of unknown origin is observed migrating at ~75 kDa. CD33 Antibody recognizes endogenous levels of total CD33 protein. Syk (D3Z1E) XP® Rabbit mAb recognizes endogenous levels of total Syk protein. Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb detects endogenous levels of Syk protein only when phosphorylated at Tyr525/526 of human Syk or Tyr519/520 of mouse Syk. It also detects Syk protein when singly phosphorylated at Tyr526 of human Syk or Tyr520 of mouse Syk. It does not cross-react with other tyrosine-phosphorylated protein tyrosine kinases. DAP12 (E7U7T) Rabbit mAb recognizes endogenous levels of total DAP12 protein.


Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu221 of human TREM2 protein, Pro110 of human DAP12 protein, Asn463 of human Syk protein, a synthetic phosphopeptide corresponding to residues surrounding Tyr525/526 of human Syk, and a recombinant protein specific to the amino terminus of human TREM2 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human CD33 protein. Antibodies are purified by peptide affinity chromatography.

Background

Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases worldwide. Clinically, it is characterized by the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles, resulting in neuronal dysfunction and cell death. Triggering receptor expressed on myeloid cells 2 (TREM2), a protein localized at the membrane of innate immune cells, including microglia in the brain, has been genetically linked to AD, with specific variants increasing disease risk by as much as threefold (1,2). The TREM2 receptor is a single-pass type I membrane glycoprotein that consists of an extracellular immunoglobulin-like domain, a transmembrane domain, and a cytoplasmic tail. Upon activation, TREM2 interacts with the tyrosine kinase-binding protein DNAX-activating protein 12 (DAP12, TYROBP) to form a receptor-signaling complex. The DAP12 protein structure consists of a short extracellular domain, a transmembrane domain, and a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) (2-9). ITAMs function as a binding site for tyrosine kinases, including spleen tyrosine kinase (Syk). Syk is comprised of two tandem amino-terminal Src homology (SH) 2 domains separated by an SH2-kinase linker, and a C-terminal tyrosine kinase domain, separated from the SH2 domains by an inter-domain linker. When Syk binds to an ITAM, it changes conformation, allowing for residues within the inter-domain linker region, including Tyr352, to become phosphorylated. Residues within the activation loop subsequently become phosphorylated, leading to full Syk activation. Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain and phosphorylation at these residues (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (10-12). This activation can lead to the mediation of a variety of cellular responses, including proliferation, differentiation, inflammation, and phagocytosis. Evidence suggests that TREM2 and DAP12 may act in a Syk-dependent manner to drive microglial cellular responses in AD (2,4-8,13).
There is also evidence that these processes may be regulated via crosstalk between TREM2 and the cell surface receptor CD33, a sialic acid-binding Ig-like lectin (Siglec-3) type I transmembrane protein. Much like TREM2, CD33 has been identified as a risk gene in AD. CD33 binds preferentially to alpha-2, 6-linked sialic acid, which can be found in sialylated gangliosides in the brain. Activation of CD33 has been shown to be inhibitory to a variety of cellular processes. Evidence suggests that TREM2 may act downstream of CD33 and that TREM2-dependent microglial signaling in AD may be directly inhibited by CD33 activation (14-17).

  1. Nguyen, A.T. et al. (2020) Acta Neuropathol 140, 477-493.
  2. Gratuze, M. et al. (2018) Mol Neurodegener 13, 66.
  3. Jonsson, T. et al. (2013) N Engl J Med 368, 107-16.
  4. Jay, T.R. et al. (2017) Mol Neurodegener 12, 56.
  5. McQuade, A. et al. (2020) Nat Commun 11, 5370.
  6. Schlepckow, K. et al. (2020) EMBO Mol Med 12, e11227.
  7. Zhao, Y. et al. (2018) Neuron 97, 1023-1031.e7.
  8. Colonna, M. (2003) Nat Rev Immunol 3, 445-53.
  9. Lanier, L.L. et al. (1998) Nature 391, 703-7.
  10. Zhang, J. et al. (2000) J Biol Chem 275, 35442-7.
  11. Mansueto, M.S. et al. (2019) J Biol Chem 294, 7658-7668.
  12. Grädler, U. et al. (2013) J Mol Biol 425, 309-33.
  13. Turner, M. et al. (2000) Immunol Today 21, 148-54.
  14. Karch, C.M. et al. (2012) PLoS One 7, e50976.
  15. Griciuc, A. et al. (2013) Neuron 78, 631-43.
  16. Griciuc, A. et al. (2019) Neuron 103, 820-835.e7.
  17. Salminen, A. et al. (2021) Neurochem Int 150, 105186.

Pathways

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