MYD88 Antibody

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ProSci
Product Code: PSI-2125
Product Group: Primary Antibodies
Supplier: ProSci
CodeSizePrice
PSI-2125-0.02mg0.02mg£150.00
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PSI-2125-0.1mg0.1mg£449.00
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Overview

Host Type: Rabbit
Antibody Isotype: IgG
Antibody Clonality: Polyclonal
Regulatory Status: RUO
Target Species:
  • Human
  • Mouse
  • Rat
Applications:
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Immunofluorescence (IF)
  • Immunohistochemistry (IHC)
  • Western Blot (WB)

Images

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<strong>Figure 1 Western Blot Validation of MyD88 in HeLa (A) and Jurket (B) Cells</strong><br> Loading: 15 μg of lysates per lane. Antibodies: 2125 (1 μg/mL) 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: MyD88 2125 (2 μg/mL), MyD88 2127 (2 μg/mL), beta-actin (1 μg/mL), and GAPDH (0.02 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 3 Independent Antibody Validation (IAV) via Protein Expression Profile in Human Tissues</strong><br> Loading: 15 μg of lysates per lane. Antibodies: MyD88 2125 (2 μg/mL), MyD88 2127 (2 μg/mL), beta-actin (1 μg/mL), and GAPDH (0.02 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 4 Animal Species Reactivity</strong><br> Loading: Lysates/proteins at 15 μg per lane. Antibodies: 2125 (2 μg/mL) or 2127 (2 μg/mL). 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 5 Validation with MyD88 siRNA Knockdown in HeLa Cells</strong><br> HeLa cells were transfected with control siRNAs (lane 1) or MyD88 siRNAs (lane 2) Loading: 10 μg of HeLa whole cell lysates per lane. Antibodies: 2125 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 6 Immunofluorescence Validation of MyD88 n Human Testis</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed human testis tissue labeling MyD88 with 2125 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing nucleus staining on human testis cells.
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<strong>Figure 7 Immunohistochemistry Validation of MyD88 in Human Heart</strong><br> Immunohistochemical analysis of paraffin-embedded human heart tissue using anti-MyD88 antibody (2125) at 2 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
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<strong>Figure 8 Immunofluorescence Validation of MyD88 in K562 Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling MyD88 with 2125 at 10 μg/mL, followed by Goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
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<strong>Figure 9 KO Validation in Mouse Macrophages (Miller et al., 2006) </strong><br> Bone marrow-derived macrophages from wild type (WT) mice and MyD88 knockout mice were assessed for MyD88 protein expression by anti-MyD88 antibodies. MyD88 expression was detected in WT mice, but not in MyD88 knockout mice.
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<strong>Figure 10 KO Validation in MyD88-deficient MEF cell line (Burns et al., 2003) </strong><br> MyD88-/- deficient MEF cell line was reconstituted by retroviral infection with an empty vector, MyD88, or MyD88s expression vectors. The levels of MyD88 (isoform 1) or MyD88s (isoform 3) were confirmed with anti-MyD88 antibodies and MyD88 expression was not detected in the MyD88-deficient cells.
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<strong>Figure 11 KO Validation in Mouse IECs (Vlantis et al., 2014) </strong><br> Western blot with anti-MyD88 antibodies on intestinal epithelial cells (IECs) showing efficient deletion of MyD88 and concomitant expression of GFP in MyD88IEC-KO mice, but not in MyD88 knockout mice.
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<strong>Figure 12 KD Validation in Chondrocytes (Ahmad et al., 2009) </strong><br> Chondrocytes were transfected with either MyD88 siRNA or control siRNA and analyzed for MyD88 expression by immunoblotting with anti-Myd88 antibodies that confirmed inhibition of the target proteins.
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<strong>Figure 13 Immunoprecipitation Validation in HEK293 cells (Kawai et al., 2004) </strong><br> HEK293 cells were transiently transfected with FLAG-IRF7. Cell lysates were immunoprecipitated with control rabbit anti-mouse immunoglobulin serum (IgG) or anti-MyD88 (Ab1 and Ab2), followed by immunoblotting with anti-FLAG.
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<strong>Figure 1 Western Blot Validation of MyD88 in HeLa (A) and Jurket (B) Cells</strong><br> Loading: 15 μg of lysates per lane. Antibodies: 2125 (1 μg/mL) 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: MyD88 2125 (2 μg/mL), MyD88 2127 (2 μg/mL), beta-actin (1 μg/mL), and GAPDH (0.02 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 Independent Antibody Validation (IAV) via Protein Expression Profile in Human Tissues</strong><br> Loading: 15 μg of lysates per lane. Antibodies: MyD88 2125 (2 μg/mL), MyD88 2127 (2 μg/mL), beta-actin (1 μg/mL), and GAPDH (0.02 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Animal Species Reactivity</strong><br> Loading: Lysates/proteins at 15 μg per lane. Antibodies: 2125 (2 μg/mL) or 2127 (2 μg/mL). 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Validation with MyD88 siRNA Knockdown in HeLa Cells</strong><br> HeLa cells were transfected with control siRNAs (lane 1) or MyD88 siRNAs (lane 2) Loading: 10 μg of HeLa whole cell lysates per lane. Antibodies: 2125 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 6 Immunofluorescence Validation of MyD88 n Human Testis</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed human testis tissue labeling MyD88 with 2125 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing nucleus staining on human testis cells.
<strong>Figure 7 Immunohistochemistry Validation of MyD88 in Human Heart</strong><br> Immunohistochemical analysis of paraffin-embedded human heart tissue using anti-MyD88 antibody (2125) at 2 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 8 Immunofluorescence Validation of MyD88 in K562 Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling MyD88 with 2125 at 10 μg/mL, followed by Goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
<strong>Figure 9 KO Validation in Mouse Macrophages (Miller et al., 2006) </strong><br> Bone marrow-derived macrophages from wild type (WT) mice and MyD88 knockout mice were assessed for MyD88 protein expression by anti-MyD88 antibodies. MyD88 expression was detected in WT mice, but not in MyD88 knockout mice.
<strong>Figure 10 KO Validation in MyD88-deficient MEF cell line (Burns et al., 2003) </strong><br> MyD88-/- deficient MEF cell line was reconstituted by retroviral infection with an empty vector, MyD88, or MyD88s expression vectors. The levels of MyD88 (isoform 1) or MyD88s (isoform 3) were confirmed with anti-MyD88 antibodies and MyD88 expression was not detected in the MyD88-deficient cells.
<strong>Figure 11 KO Validation in Mouse IECs (Vlantis et al., 2014) </strong><br> Western blot with anti-MyD88 antibodies on intestinal epithelial cells (IECs) showing efficient deletion of MyD88 and concomitant expression of GFP in MyD88IEC-KO mice, but not in MyD88 knockout mice.
<strong>Figure 12 KD Validation in Chondrocytes (Ahmad et al., 2009) </strong><br> Chondrocytes were transfected with either MyD88 siRNA or control siRNA and analyzed for MyD88 expression by immunoblotting with anti-Myd88 antibodies that confirmed inhibition of the target proteins.
<strong>Figure 13 Immunoprecipitation Validation in HEK293 cells (Kawai et al., 2004) </strong><br> HEK293 cells were transiently transfected with FLAG-IRF7. Cell lysates were immunoprecipitated with control rabbit anti-mouse immunoglobulin serum (IgG) or anti-MyD88 (Ab1 and Ab2), followed by immunoblotting with anti-FLAG.

Documents

Further Information

Additional Names:
MYD88 Antibody: Myeloid differentiation primary response 88, MYD88D
Application Note:
WB: 1 - 2 μg/mL; IHC-P: 2 μg/mL; IF: 10-20 μg/mL.

Antibody validated: Western Blot in human, mouse and rat samples; Immunohistochemistry, Immunoprecipitation and Immunofluorescence in human samples. All other applications and species not yet tested.
Background:
MYD88, myeloid differentiation primary response 88, was identified as an innate immune signal transduction adaptor involved in the Toll-like receptor (TLR) and interleukin-1 (IL-1) signaling pathway (1,2,3) and plays an important role in the inflammatory response induced by cytokines IL-1 and IL-18 and endotoxin. MyD88 functions as an adaptor protein for TLRs and IL-1 receptors, which stimulates IRAKs, IRF7 and TRAF6, leading to NF-?B activation, cytokine secretion and inflammatory response (2, 4,5,6). Nuclear factor-kappa-B activation modulates multiple genes regulating the body's immune reactions and inflammatory responses. MyD88 associates with and recruits IRAK to the IL-1 receptor complex in response to IL-1 treatment and dominant negative form of MyD88 attenuates IL-1R-mediated NF-?B activation(4,5). MyD88 is also employed as a regulator molecule by IL-18 receptor. Targeted disruption of the MyD88 gene results in loss of cellular responses to IL-1 and IL-18, and MyD88-deficient mice lack responses to bacterial product LPS that employs TLR2 and TLR4 as the signaling receptors(7,8). MyD88 gene is expressed in many tissues.
Background References:
  • Medzhitov et al. Mol. Cell. 1998;2:253-8
  • Kawai et al. Nat. Immunol. 2004;5:1061-8
  • Semaan et al. J. Immunol. 2008;180:3485-91
  • Muzio et al. Science 1997;278:1612-5
  • Yamamoto et al. Mol. Immunol. 2014;58:66-76
  • Ohnishi et al. Proc. Natl. Acad. Sci. U.S.A. 2009;106:10260-5
  • Adachi et al. Immunity 1998;9:143-50
  • Kawai et al. Immunity 1999;11:115-22
Buffer:
MYD88 Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Conjugate:
Unconjugated
DISCLAIMER:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.
Homology:
Predicted species reactivity based on immunogen sequence: Pig: (100%), Sheep: (100%), Bovine: (100%), Chicken: (100%)
Immunogen:
Anti-MYD88 antibody (2125) was raised against a peptide corresponding to 16 amino acids near the center of human MYD88 isoform 1.

The immunogen is located within amino acids 220 - 270 of MYD88.
ISOFORMS:
Human MYD88 has 7 isoforms, including isoform 1 (317aa, 35.4kD), isoform 2 (296aa, 33.2 kD), isoform 3 (251aa, 28.3kD), isoform 4 (191aa, 20.8kD), isoform 5 (146aa, 15.8kD), isoform 6 (275aa, 31.5kD), and isoform 7 (304aa, 34.1kD). This antibody detects human isoform 1,2,3,6,7, but not isoform 4,5. Mouse MYD88 has two isoforms, including isoform 1 (296aa, 33.8kD) and isoform 2 (250aa, 28.7kD). Rat MYD88 has only one isoform identified so far (296aa, 33.9kD).
NCBI Gene ID #:
4615
NCBI Official Name:
myeloid differentiation primary response gene (88)
NCBI Official Symbol:
MYD88
NCBI Organism:
Homo sapiens
Physical State:
Liquid
PREDICTED MOLECULAR WEIGHT:
Predicted: 35kD

Observed: 35kD
Protein Accession #:
AAB49967.1
Protein GI Number:
1763090
Purification:
MYD88 Antibody is affinity chromatography purified via peptide column.
Research Area:
Signal Transduction,Innate Immunity
Swissprot #:
U70451
User NOte:
Optimal dilutions for each application to be determined by the researcher.
VALIDATION:

Independent Antibody Validation in Cell lines (Figure 2) shows similar MYD88 expression profile in both human and mouse cell lines detected by two independent anti-MYD88 antibodies that recognize different epitopes, 2125 against internal domain and 2127 against the C-terminus domain.  MYD88 proteins are detected in all the tested cell lines at different expression levels by the two independent antibodies.  Additionally, Figure 2 shows the mouse MYD88 protein in NIH/3T3 cells migrates slightly faster than human isoform 1 detected by both MYD88 antibodies (2125 and 2127), which is well correlated with their calculated molecular masses (33.8 kDa vs 35.4 kDa).  

Independent Antibody Validation in Human Tissues (Figure 3) shows similar MYD88 expression profile in human tissues detected by two independent anti-MYD88 antibodies (2125 and 2127). MYD88 proteins are detected by the two independent antibodies in liver, kidney, lung, thymus, colon, bladder and breast of human tissues at different expression levels, but not in heart, brain, skin and pancreas.

Animal Species Reactivity (Figure 4): Anti-MYD88 antibodies (2125 and 2127) can detect the expression of MYD88 protein in the liver and spleen of all tissues and mouse heart, but not in human heart. Additionally, Figure 4 also shows MYD88 protein detected by both MYD88 antibodies (2125 and 2127) in human liver and Daudi cells migrates slightly slower than that in the tissues of mouse and rat, which is well correlated with their calculated molecular masses (35.4 kDa vs 33.8 kDa and 33.9kD). 

siRNA knockdown validation (Figure 5): Anti-MYD88 antibody (2125) specificity was further verified by MYD88 specific siRNA knockdown. MYD88 signal in HeLa cells transfected with MYD88 siRNAs was weaker in comparison with that in HeLa cells transfected with control siRNAs.

References

  1. Miller et al. MyD88 media16413925tes neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus. Immunity. 2006;24(1):79-91. PMID: 16413925
  2. Burns et al. Inhibition of Interleukin 1 Receptor/Toll-like Receptor Signaling through the Alternatively Spliced, Short Form of MyD88 Is Due to Its Failure to Recruit IRAK-4. J Exp Med. 2003;197(2): 263-8. PMID: 12538665
  3. Vlantis et al. TLR-independent anti-inflammatory function of intestinal epithelial TRAF6 signalling prevents DSS-induced colitis in mice. Gut. 2016;65(6):935-43.PMID: 25761602
  4. Ahmad et al. Elevated expression of the toll like receptors 2 and 4 in obese individuals: its significance for obesity-induced inflammation. J Inflamm (Lond). 2012;9:48. PMID: 23191980
  5. Kawai et al. Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. Nat Immunol. 2004;5(10):1061-8.PMID: 15361868
  6. Romics L Jr et al. Selective priming to Toll-like receptor 4 (TLR4), not TLR2, ligands by P. acnes involves up-regulation of MD-2 in mice. Hepatology. 2004;40(3):555-64PMID: 15349893
  7. Ahmad et al. Requirement of TLR2-mediated signaling for the induction of IL-15 gene expression in human monocytic cells by HSV-1. Blood. 2008;112(6):2360-8. PMID: 18583567
  8. Kawagoe T et al. Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor-mediated immune responses but not in TCR signaling. J Exp Med. 2007;204(5):1013-24. PMID: 17485511
  9. Lin et al. The tyrosine kinase Syk differentially regulates Toll-like receptor signaling downstream of the adaptor molecules TRAF6 and TRAF3. Sci Signal. 2013;6(289):ra71. PMID: 23962979
  10. Ahmad et al. MyD88, IRAK1 and TRAF6 knockdown in human chondrocytes inhibits interleukin-1-induced matrix metalloproteinase-13 gene expression and promoter activity by impairing MAP kinase activation. Cell Signal. 2007;19(12):2549-57. PMID: 17905570
  11. Mukherjee et al. Lipopolysaccharide-driven Th2 Cytokine Production in Macrophages Is Regulated by Both MyD88 and TRAM. J Biol Chem. 2009;284(43): 29391-8.PMID: 19638630
  12. Campanholle et al. TLR-2/TLR-4 TREM-1 Signaling Pathway Is Dispensable in Inflammatory Myeloid Cells during Sterile Kidney Injury. PLoS One. 2013;8(7): e68640. PMID: 23844229
  13. Murakami et al. Human herpesvirus 6 infection impairs Toll-like receptor signaling. Virol J. 2010;7:91.PMID: 20459723
  14. Su et al. Expression and regulation of Toll-like receptor 2 by IL-1beta and fibronectin fragments in human articular chondrocytes. Osteoarthritis Cartilage. 2005;13(10):879-86. PMID: 15961329
  15. Pedrosa et al. Bacteria and spontaneous experimental colitis: immunological changes. Eur J Clin Invest. 2011;41(10):1047-53. PMID: 21902690

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