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98110
Necroptosis Antibody Sampler Kit

Necroptosis Antibody Sampler Kit #98110

Western Blotting Image 1

Western blot analysis of extracts from various cell lines using RIP (D94C12) XP® Rabbit mAb.

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Western Blotting Image 2

Western blot analysis of HT-29 cells, untreated (-) or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP (Ser166) (D1L3S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 3

Western blot analysis of extracts from various cell lines using MLKL (D2I6N) Rabbit mAb. KARPAS cell Line source: Dr. Abraham Karpas at the University of Cambridge.

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Western Blotting Image 4

Western blot analysis of HT-29 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb (upper), or MLKL (D2I6N) Rabbit mAb #14993 (lower).

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using RIP3 (E1Z1D) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 6

Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb. To confirm phospho-specificity, membranes were either untreated (left) or treated with Calf Intestinal Phosphatase (CIP; right).

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Western Blotting Image 7

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.

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Western Blotting Image 8

Western blot analysis of extracts from HeLa cells, untransfected or transfected with human RIP construct, using RIP (D94C12) XP® Rabbit mAb.

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Western Blotting Image 9

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human MLKL protein (hMLKL-Myc/DDK; +), using MLKL (D2I6N) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).

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Western Blotting Image 10

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human RIP3 protein (hRIP3; +), using RIP3 (E1Z1D) Rabbit mAb.

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Western Blotting Image 11

Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Flow Cytometry Image 12

Flow cytometric analysis of MCF7 cells using RIP (D94C12) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).

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Western Blotting Image 13

Western blot analysis of HT-29 cells or HT-29 RIPK1 KO cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle) or β-Actin (D6A8) Rabbit mAb #8457 (lower). HT-29 RIPK1 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.

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IF-IC Image 14

Confocal immunofluorescent analysis of OVCAR8 cells using RIP (D94C12) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-IC Image 15

Confocal immunofluorescent analysis of HT-29 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 6 hr; center), or pre-treated with Z-VAD followed by treatment with SM-164 and hTNF-α and post-processed with λ-phosphatase (right), using Phospho-RIPK3 (Ser227) (D2W2T) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
RIP (D94C12) XP® Rabbit mAb 3493 20 µl
  • WB
  • IP
  • IF
  • F
H M R Hm Mk 78 Rabbit IgG
Phospho-RIP (Ser166) (D1L3S) Rabbit mAb 65746 20 µl
  • WB
H 78-82 Rabbit IgG
MLKL (D2I6N) Rabbit mAb 14993 20 µl
  • WB
H 54 Rabbit IgG
Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb 91689 20 µl
  • WB
H 54 Rabbit IgG
RIP3 (E1Z1D) Rabbit mAb 13526 20 µl
  • WB
  • IP
H 46-62 Rabbit IgG
Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb 93654 20 µl
  • WB
  • IF
H 46-62 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Necroptosis Antibody Sampler Kit provides an economical means of detecting total and phosphorylated proteins associated with necroptosis. The kit includes enough antibody to perform two western blots with each primary antibody.

Each antibody in the Necroptosis Antibody Sampler Kit detects endogenous levels of its target protein. MLKL (D2I6N) Rabbit mAb cross-reacts with an unidentified band at 130 kDa in some cell lines. Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb may also bind to MLKL when dually phosphorylated at Thr357 and Ser358.

Monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to Leu190 of human RIP, residues near the carboxyl terminus of human RIP3, and residues near the carboxyl terminus of human MLKL. Phospho-specific monoclonal antibodies are produced by immunizing rabbits with synthetic phospho-peptides corresponding to Ser166 of human RIP, Ser227 of human RIP3, and Ser358 of human MLKL.

Necroptosis, a regulated pathway for necrotic cell death, is triggered by a number of inflammatory signals, including cytokines in the tumor necrosis factor (TNF) family, pathogen sensors such as toll-like receptors (TLRs), ischemic injury, and neurodegenerative diseases (1-3). The process is negatively regulated by caspases and is initiated through a complex containing the RIP and RIP3 kinases, typically referred to as the necrosome. Necroptosis is inhibited by a small molecule inhibitor of RIP, necrostatin-1 (Nec-1) (4). RIP is phosphorylated at several sites within the kinase domain that are sensitive to Nec-1, including Ser14, Ser15, Ser161, and Ser166 (5). During necroptosis, RIP3 is phosphorylated at Ser227, leading to recruitment and phosphorylation of MLKL at Thr357 and Ser358 (6). Phosphorylation of MLKL results in its oligomerization and translocation to the plasma membrane, where it effects membrane integrity (7-10).

  1. Christofferson, D.E. and Yuan, J. (2010) Curr Opin Cell Biol 22, 263-8.
  2. Kaczmarek, A. et al. (2013) Immunity 38, 209-23.
  3. Zhou, W. and Yuan, J. (2014) Semin Cell Dev Biol 35, 14-23.
  4. Degterev, A. et al. (2008) Nat Chem Biol 4, 313-21.
  5. Ofengeim, D. and Yuan, J. (2013) Nat Rev Mol Cell Biol 14, 727-36.
  6. Sun, L. et al. (2012) Cell 148, 213-27.
  7. Cai, Z. et al. (2014) Nat Cell Biol 16, 55-65.
  8. Chen, X. et al. (2014) Cell Res 24, 105-21.
  9. Wang, H. et al. (2014) Mol Cell 54, 133-46.
  10. Dondelinger, Y. et al. (2014) Cell Rep 7, 971-81.
Entrez-Gene Id
197259 , 8737 , 11035
Swiss-Prot Acc.
Q8NB16 , Q13546 , Q9Y572
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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