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90144
NETosis Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit
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NETosis Antibody Sampler Kit #90144

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NETosis Antibody Sampler Kit: Image 1
Western blot analysis of extracts from various cell lines using Myeloperoxidase (E1E7I) XP® Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
NETosis Antibody Sampler Kit: Image 2
Western blot analysis of extracts from various cell lines using Histone H3 (D1H2) XP® Rabbit mAb.
NETosis Antibody Sampler Kit: Image 3
Western blot analysis of extracts from U-937, THP-1, and HuH-6 cells, untreated (-) or treated with PNGase F (+), using Cathepsin G (E3N3O) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The lack of detectable Cathepsin G expression in HuH-6 cells is consistent with proteomic and transcriptomic expression profiling data, confirming specificity of the antibody.
NETosis Antibody Sampler Kit: Image 4
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.
NETosis Antibody Sampler Kit: Image 5
Western blot analysis of extracts from various cell lines using Neutrophil Elastase (E9C9L) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, neutrophil elastase protein expression is not detected in either A-498 or Jurkat cells.
NETosis Antibody Sampler Kit: Image 6
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing human PADI4 (hPADI4; +), and untreated (-) or treated (+) as indicated with: Locke’s Solution (30 min) and A23187 ionophore (5 μM, 30 min), using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). Citrullination of histone H3 is induced by expression of PADI4, along with activation by Locke’s Solution and A23187 ionophore treatment, as expected.
NETosis Antibody Sampler Kit: Image 7
Immunohistochemical analysis of paraffin-embedded human pulmonary sarcoma using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 8
Immunohistochemical analysis of paraffin-embedded colon adenocarcinoma using Myeloperoxidase (E1E7I) XP® Rabbit mAb.
NETosis Antibody Sampler Kit: Image 9
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Histone H3 (D1H2) XP® Rabbit mAb.
NETosis Antibody Sampler Kit: Image 10
Immunohistochemical analysis of paraffin-embedded human esophageal adenocarcinoma using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 11
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human neutrophil elastase protein (hNE-Myc/DDK; +), using Neutrophil Elastase (E9C9L) XP® Rabbit mAb (upper), DYKDDDDK Tag Antibody #2368 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
NETosis Antibody Sampler Kit: Image 12
Immunohistochemical analysis of paraffin-embedded human mucoepidermoid carcinoma of the larynx using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 13
Immunohistochemical analysis of paraffin-embedded non-small cell lung carcinoma using Myeloperoxidase (E1E7I) XP® Rabbit mAb.
NETosis Antibody Sampler Kit: Image 14
Confocal immunofluorescent analysis of HeLa cells using Histone H3 (D1H2) XP® Rabbit mAb (green) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red).
NETosis Antibody Sampler Kit: Image 15
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Cathepsin G (E3N3O) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
NETosis Antibody Sampler Kit: Image 16
Immunoprecipitation of neutrophil elastase 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 Neutrophil Elastase (E9C9L) XP® Rabbit mAb. Western blot analysis was performed using Neutrophil Elastase (E9C9L) XP® Rabbit mAb. Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used for detection to avoid cross-reactivity with IgG.
NETosis Antibody Sampler Kit: Image 17
Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 18
Immunohistochemical analysis of paraffin-embedded HL-60 (left) and HeLa (right) cell pellets using Myeloperoxidase (E1E7I) XP® Rabbit mAb.
NETosis Antibody Sampler Kit: Image 19
Flow cytometric analysis of HeLa cells using Histone H3 (D1H2) XP® Rabbit mAb (solid line) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
NETosis Antibody Sampler Kit: Image 20
Immunohistochemical analysis of paraffin-embedded THP-1 cell pellet (left, positive) or HuH-6 cell pellet (right, negative) using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 21
Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA #4174 (80 nM, 24 hr; left, positive) and A-498 cells (right, negative), using Neutrophil Elastase (E9C9L) XP® Rabbit mAb (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
NETosis Antibody Sampler Kit: Image 22
Immunohistochemical analysis of paraffin-embedded normal human spleen using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 23
Confocal immunofluorescent analysis of HL-60 cells (left, positive) or HeLa cells (right, negative) using Myeloperoxidase (E1E7I) XP® Rabbit mAb #14569 (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
NETosis Antibody Sampler Kit: Image 24
Immunohistochemical analysis of paraffin-embedded normal human colon using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 25
Flow cytometric analysis of Jurkat cells (blue) and U-937 cells (green) using Neutrophil Elastase (E9C9L) XP® Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
NETosis Antibody Sampler Kit: Image 26
Immunohistochemical analysis of paraffin-embedded normal human thymus using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 27
Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 28
Immunohistochemical analysis of paraffin-embedded 4T1 syngeneic tumor using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 29
Immunohistochemical analysis of paraffin-embedded normal human lymph node using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 30
Immunohistochemical analysis of paraffin-embedded MC38 syngeneic tumor using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 31
Immunohistochemical analysis of paraffin-embedded human non-Hodgkin lymphoma using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 32
Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb in the presence of non-citrullinated peptide (left) or Arg17 citrullinated peptide (right).
NETosis Antibody Sampler Kit: Image 33
Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 34
Immunohistochemical analysis of paraffin-embedded Renca syngeneic tumor using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right). 
NETosis Antibody Sampler Kit: Image 35
Flow cytometric analysis of HeLa cells (blue, negative) and HL-60 (green, positive) using Myeloperoxidase (E1E7I) XP® Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
NETosis Antibody Sampler Kit: Image 36
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 37
Immunohistochemical analysis of paraffin-embedded 293T cell pellets transfected with a construct expressing human PADI4 (hPADI4), untreated (left) or treated with Locke’s Solution (30 min) and A23187 ionophore (5 μM, 30 min) (right) using Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 38
Immunohistochemical analysis of paraffin-embedded normal human spleen using Cathepsin G (E3N3O) Rabbit mAb.
NETosis Antibody Sampler Kit: Image 39
Immunohistochemical analysis of paraffin-embedded human gastric carcinoma using Cathepsin G (E3N3O) Rabbit mAb.
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To Purchase # 90144
Cat. # Size Qty. Price
90144T		 1 Kit  (5 x 20 microliters)
$ 455

Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Neutrophil Elastase (E9C9L) XP® Rabbit mAb 89241 20 µl
  • WB
  • IP
  • IF
  • F
 H 30 Rabbit IgG
Myeloperoxidase (E1E7I) XP® Rabbit mAb 14569 20 µl
  • WB
  • IHC
  • IF
  • F
 H 60, 80-90 Rabbit IgG
Histone H3 (D1H2) XP® Rabbit mAb 4499 20 µl
  • WB
  • IHC
  • IF
  • F
 H M R Mk 17 Rabbit IgG
Cathepsin G (E3N3O) Rabbit mAb 63665 20 µl
  • WB
  • IHC
 H 29 Rabbit IgG
Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb 97272 20 µl
  • WB
  • IHC
 H M 17 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Rab Goat 

Product Description

The NETosis Antibody Sampler Kit provides an economical means of detecting proteins involved in NETosis. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the NETosis Antibody Sampler Kit detects endogenous levels of its target protein. Myeloperoxidase (E1E7I) XP® Rabbit mAb recognizes the full-length and heavy chain subunits of human myeloperoxidase protein. Citrullinated Histone H3 (Arg17) (E4O3F) Rabbit mAb may also recognize histone H3 citrullinated at residue Arg26 but does not cross-react with any other known citrullinated or methylated arginine residues on histone H3. This antibody may react with a band of unknown identity at 38 kDa. Histone H3 (D1H2) XP® Rabbit mAb detects isoforms H3.1, H3.2, and H3.3. This antibody also detects the histone H3 variant CENP-A. This antibody does not cross-react with other core histones.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Pro734 of human myeloperoxidase protein, Pro130 of human Cathepsin G protein, residues near the carboxy terminus of human neutrophil elastase protein and histone H3, and residues near the amino terminus of histone H3 in which Arg17 is citrullinated.

Background

NETosis is a unique form of regulated cell death that is characterized by membrane rupture and the extrusion of chromatin, histones, and granular and cytoplasmic components into a web-like structure called neutrophil extracellular traps (NETs) (reviewed in 1). NETosis has been associated with host defense to pathogens as well as a number of disease states, including autoimmune diseases, thrombosis, cardiovascular diseases, and tumor progression. NETosis was identified as a response to bacterial infection and can be activated by lipopolysaccharide (LPS) as well as inflammatory pathway activators like phorbol-12-myristate-13-acetate (PMA) (2). It can occur via multiple pathways, but several key players have emerged. The calcium-dependent enzyme protein-arginine deiminase 4 (PAD4) catalyzes hypercitrullination of histones that contributes to chromatin decondensation (3-5). In addition, activation of proteases, including neutrophil elastase (ELANE), myeloperoxidase (MPO), and Cathepsin G, leads to impairment of cytoskeletal structures and degradation of histones during NETosis (6,7).



  1. Thiam, H.R. et al. (2020) Annu Rev Cell Dev Biol 36, 191-218.
  2. Brinkmann, V. et al. (2004) Science 303, 1532-5.
  3. Li, P. et al. (2010) J Exp Med 207, 1853-62.
  4. Wong, S.L. and Wagner, D.D. (2018) FASEB J, fj201800691R.
  5. Papayannopoulos, V. et al. (2010) J Cell Biol 191, 677-91.
  6. Metzler, K.D. et al. (2014) Cell Rep 8, 883-96.

Pathways

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Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
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