MYD88 Antibody

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ProSci
Product Code: PSI-2127
Product Group: Primary Antibodies
Supplier: ProSci
CodeSizePrice
PSI-2127-0.02mg0.02mg£150.00
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PSI-2127-0.1mg0.1mg£449.00
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Overview

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

Images

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<strong>Figure 1 KO Validation in HeLa Cells</strong><br> Loading: 10 μg of HeLa WT cell lysate or MyD88 KO cell lysate. Antibodies: MyD88 2127 (2 μg/mL) and beta-actin 3779 (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 3 Western Blot Validation of MyD88 in human cell lines</strong><br>Loading: 15 μg of lysates per lane.Antibodies: 2127 (2 μg/mL) 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.Predicted band size: 35 kDa
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<strong>Figure 4 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 5 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 6 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 7 Immunofluorescence Validation of MyD88 in Jurkat Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed Jurkat cells labeling MyD88 with 2127 at 20 μg/mL followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
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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 2127 at 20 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 of MyD88 in Mouse T cells (Rahman et al. 2011) </strong><br>Splenocytes were isolated from a <em>Myd88</em>Δ<em>T</em> mouse in which MyD88 was specifically disrupted in T cells. T and B cells were FACS purified and MyD88 expression was examined by Western blot with anti-MyD88 antibodies (2127). MyD88 expression was detected in Splenocytes and B cells but not in T cells.
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<strong>Figure 10 KO Validation in CD45 <sup>-</sup> Thy1 <sup>+</sup> cells (Kim et al. 2017) </strong><br>The expression of Myd88 protein was analyzed in CD45 <sup>-</sup> Thy1 <sup>+</sup> intestinal mesenchymal cells and CD3 <sup>+</sup> intestinalT cells by immunoblotting with anti-Myd88 antibodies (2127). MyD88 expression was not detected in knockout cells.
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<strong>Figure 11 KD Validation in Raw 264.7 cells (Altimeier et al. 2007) </strong><br>The Transfection of RAW 264.7 cells with MyD88-specific siRNAresulted in attenuation of MyD88 protein by Western blot analysis with anti-Myd88 antibodies (2127).
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<strong>Figure 12 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 2. Immunofluorescence Validation of MyD88 In HeLa Cells</strong><br> Immunofluorescent analysis of methanol-fixed HeLa cells labeling MyD88 with 2127 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/1000 dilution (red) and Hoechst staining (blue). Alpha tubulin was stained with anti-alpha tubulin antibody following by goat anti-mouse IgG secondary antibody (green). Images were captured with confocal microscopy.
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<strong>Figure 1 KO Validation in HeLa Cells</strong><br> Loading: 10 μg of HeLa WT cell lysate or MyD88 KO cell lysate. Antibodies: MyD88 2127 (2 μg/mL) and beta-actin 3779 (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 3 Western Blot Validation of MyD88 in human cell lines</strong><br>Loading: 15 μg of lysates per lane.Antibodies: 2127 (2 μg/mL) 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.Predicted band size: 35 kDa
<strong>Figure 4 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 5 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 6 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 7 Immunofluorescence Validation of MyD88 in Jurkat Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed Jurkat cells labeling MyD88 with 2127 at 20 μg/mL followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
<strong>Figure 8 Immunofluorescence Validation of MyD88 in K562 Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling MyD88 with 2127 at 20 μg/mL followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
<strong>Figure 9 KO Validation of MyD88 in Mouse T cells (Rahman et al. 2011) </strong><br>Splenocytes were isolated from a <em>Myd88</em>Δ<em>T</em> mouse in which MyD88 was specifically disrupted in T cells. T and B cells were FACS purified and MyD88 expression was examined by Western blot with anti-MyD88 antibodies (2127). MyD88 expression was detected in Splenocytes and B cells but not in T cells.
<strong>Figure 10 KO Validation in CD45 <sup>-</sup> Thy1 <sup>+</sup> cells (Kim et al. 2017) </strong><br>The expression of Myd88 protein was analyzed in CD45 <sup>-</sup> Thy1 <sup>+</sup> intestinal mesenchymal cells and CD3 <sup>+</sup> intestinalT cells by immunoblotting with anti-Myd88 antibodies (2127). MyD88 expression was not detected in knockout cells.
<strong>Figure 11 KD Validation in Raw 264.7 cells (Altimeier et al. 2007) </strong><br>The Transfection of RAW 264.7 cells with MyD88-specific siRNAresulted in attenuation of MyD88 protein by Western blot analysis with anti-Myd88 antibodies (2127).
<strong>Figure 12 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.
<strong>Figure 2. Immunofluorescence Validation of MyD88 In HeLa Cells</strong><br> Immunofluorescent analysis of methanol-fixed HeLa cells labeling MyD88 with 2127 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/1000 dilution (red) and Hoechst staining (blue). Alpha tubulin was stained with anti-alpha tubulin antibody following by goat anti-mouse IgG secondary antibody (green). Images were captured with confocal microscopy.

Documents

Further Information

Additional Names:
MYD88 Antibody: MYD88D
Application Note:
WB: 2 μg/mL; IF: 20 μg/mL.

Antibody validated: Western Blot in human, mouse and rat samples; Immunofluorescence and Immunoprecipitation 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: (94%), Sheep: (82%), Bovine: (82%), Chicken: (82%)
Immunogen:
Anti-MYD88 antibody (2127) was raised against a peptide corresponding to 17 amino acids near carboxy terminus of human MYD88 isoform 1.

The immunogen is located within the last 50 amino acids 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
Swissprot #:
U70451
User NOte:
Optimal dilutions for each application to be determined by the researcher.
VALIDATION:

KO validation (Figure 1): Anti-MYD88 antibody (2127) specificity was further verified by MYD88 specific knockout. MyD88 signal was not detected in MyD88 knockout HeLa cells as compared to that in control wild type cells.

Independent Antibody Validation in Cell lines (Figure 3) 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 4) 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 5): 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). 

References

  1. Rahman et al. Antiviral memory CD8 T-cell differentiation, maintenance, and secondary expansion occur independently of MyD88. Blood. 2011; 117(11): 3123-30. PMID: 21233312
  2. Kim et al. Mesenchymal Cell-Specific MyD88 Signaling Promotes Systemic Dissemination of Salmonella Typhimurium via Inflammatory Monocytes. J Immunol. 2017; 199(4):1362-1371. PMID: 28674182
  3. Altemeier et al. Fas (CD95) Induces Macrophage Pro-Inflammatory Chemokine Production via a MyD88-dependent, Caspase-independent Pathway. J Leukoc Biol. 2007; 82(3): 721-8PMID: 17576821
  4. 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):1061-8PMID: 15361868
  5. 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):555-64. PMID: 15349893
  6. Miller et al. MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus. Immunity. 2006;24(1):79-91. PMID: 16413925
  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. 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): 263-8PMID: 12538665
  10. 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
  11. 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
  12. 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
  13. 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
  14. 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
  15. Murakami et al. Human herpesvirus 6 infection impairs Toll-like receptor signaling. Virol J. 2010;7:91. PMID: 20459723

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