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<strong>Figure 1 Western Blot Validation in Human Cell Lines</strong><br>Loading: 15 μg/ of lysates per lane.Antibodies: IRAK4 3125 (1 μg/mL), 1h 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: IRAK4-3125 (1 μg/mL), IRAK4-24-025 (1 μg/mL), beta-actin (1.5 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 Western Blot Validation in Human HeLa Cell Lines</strong><br>Loading: 15 μg of lysates per lane.Antibodies: IRAK4 3125, 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.Lane 1: 1 μg/mLLane 2: 2 μg/mLLane 3: 4 μg/mL
<strong>Figure 4 Immunocytochemistry Validation of IRAK4 in K562 Cells</strong><br>Immunocytochemical analysis of K562 cells using anti-IRAK4 antibody (3125) at 10 μg/ml. Cells 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 5 Immunofluorescence Validation of IRAK4 in K562 Cells</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 Cells labeling IRAK4 with 3125 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
<strong>Figure 6 KO and KD Validation of IRAK4 in Mouse Bone Marrow-derived Macrophages (BMDM) (Koziczak-Holbro et al., 2008) </strong><br>Western blot analysis with anti-IRAK4 antibodies was performed for IRAK4 in BMDM isolated from the mice. IRAK4 expression was not observed in the IRAK-4-/- cells and also reduced in IRAK4 mutant (IRAK-4 KD) when compared with WT.
<strong>Figure 7 Regulated Expression Validation of IRAK4 in Mouse RAW 264 Cells (Hatao et al., 2004) </strong><br>RAW 264 cells were stimulated with (a) CSK4 and (b) CpG-DNA in the absence or presence of proteasome inhibitors (MG132 and Lactactstin). When detected with anti-IRAK4 antibodies (C12 from ProSci, Inc.), IRAK4 expression was found to be reduced without the inhibitors. The smaller protein band, a cleavage product of IRAK4, was present in the absence of both inhibitors.
<strong>Figure 8 KD Validation of IRAK4 in HEK293T Cells (Heinz et al., 2012) </strong><br>Western blot analysis with anti-IRAK4 antibodies was performed for IRAK4 in HEK293T cells. IRAK4 expression was not observed in IRAK4 knockdown cells.
Figure 1 Western Blot Validation in Human Cell Lines
Loading: 15 μg/ of lysates per lane.Antibodies: IRAK4 3125 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
Cell Treatment
IAV
Trial Size

IRAK-4 Antibody

CATALOG NUMBER: 3125

Clonality:
Polyclonal
Tested Applications:
ELISA, ICC, IF, WB
Host Species:
Rabbit
Species Reactivity:
Human
Conjugate:
Unconjugated
Specifications
Host Species:
Rabbit
Species Reactivity:
Human
Homology:
Predicted species reactivity based on immunogen sequence: Bovine: (85%), Mouse: (85%), Rat (77%)
Immunogen:
Anti-IRAK4 antibody (3125) was raised against a peptide corresponding to 13 amino acids near the carboxy terminus of human IRAK4.
The immunogen is located within the last 50 amino acids of IRAK4.
Conjugate:
Unconjugated
Tested Applications:
ELISA, ICC, IF, WB
Application Note:
WB: 1-4 μg/mL; ICC: 10 μg/mL; IF: 10 μg/mL.
Antibody validated: Western Blot in human samples; Immunocytochemistry in human samples and Immunofluorescence in human samples. All other applications and species not yet tested.
Specificity:
IRAK-4 antibody is predicted to not cross-react with other members of the IRAK protein family.
Positive Control 1:
Cat. No. 1204 - K562 Cell Lysate
Positive Control 2:
Cat. No. 1201 - HeLa Cell Lysate
Positive Control 3:
Cat. No. 1224 -Daudi Cell Lysate
Positive Control 4:
Cat. No. 1211 -HepG2 Cell Lysate
Positive Control 5:
Cat. No. 17-004 - K-562 Cell Slide
Predicted Molecular Weight:
Predicted: 52kD
Observed: 55 kD
Validation:

Independent Antibody Validation in Cell lines (Figure 2) shows similar IRAK4 expression profile in human cell lines detected by two independent anti-IRAK4 antibodies that recognize different epitopes, 3125 against C-terminus domain and 24-025 against recombinant fusion protein. IRAK4 proteins are detected in the most tested cell lines at different expression levels by the two independent antibodies.

KO validation (Figure 6): Anti-IRAK4 antibody (3125) specificity was further verified by IRAK4 knockout and mutant mice. IRAK4 signal in the macrophages of IRAK KO mice and mutant mice was disrupted as compared to wild type mice.

Regulated expression validation (Figure 7): IRAK4 expression detected by anit-IRAK4 antibodies (3125) was reduced with CSK-4 treatment, which was rescued by proteasome inhibitors.

KD validation (Figure 8): Anti-IRAK4 antibody (3125) specificity was further verified by IRAK4 knockdown. IRAK4 signal was disrupted in HEK293T cells transfected with IRAK4 siRNAs.

Isoforms:
Human IRAK4 has 2 isoforms, including isoform 1 (460aa, 52kD) and isoform 2 (336aa, 38kD). Mouse IRAK4 has one isoform (459aa, 51kD) and Rat IRAK4 also has one isoform (461aa, 51kD). 3125 can detect human isoforms as well as mouse and rat IRAK4.
Purification:
IRAK-4 Antibody is affinity chromatography purified via peptide column.
Clonality:
Polyclonal
Isotype:
IgG
physical-state:
Liquid
Buffer:
IRAK-4 Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Storage Conditions:
IRAK-4 antibody can be stored at 4°C for three months and -20°C, stable for up to one year. As with all antibodies care should be taken to avoid repeated freeze thaw cycles. Antibodies should not be exposed to prolonged high temperatures.
Ncbi Official Symbol:
IRAK4
Additional Names:
IRAK-4 Antibody: IPD1, REN64, IRAK-4, NY-REN-64, Interleukin-1 receptor-associated kinase 4, Renal carcinoma antigen NY-REN-64
Protein Accession Number:
AAM15772
Protein Gi Number:
20219010
Ncbi Gene Id Number:
51135
User Note:
Optimal dilutions for each application to be determined by the researcher.
Background:
IRAK-4 Antibody: Interleukin-1 (IL-1) and lipopolysaccharide (LPS) induces cellular responses through IL-1 receptor (IL-1R) and Toll-like receptors (TLR). IL-1R-associated kinases (IRAK, IRAK2, and IRAK-M) regulate the activation of NF-κB and MAP kinase (MAPK) by IL-1R/TLR. A novel member in the IRAK/Pelle family was recently identified and designated IRAK-4. Overexpression of IRAK-4 activates NF-κB and MAPK pathways. IRAK-4 interacts with and phosphorylates IRAK-1. IRAK-4-deficient animals are completely resistant to the challenge with LPS. Animals and humans lacking IRAK-4 are impaired in their responses to viral and bacterial challenges. Members in IRAK/Pelle family play a central role in IL-1R/TLR mediated inflammatory responses and in innate immunity.
Background Reference 1:
Cao et al. Science 1996; 271:1128-31
Background Reference 2:
Muzio et al. Science 1997; 278:1612-5.
Background Reference 3:
Wesche et al. J. Biol. Chem. 1999; 274:19403-10.
Background Reference 4:
Li et al. Proc. Natl. Acad. Sci. USA 2002; 99:5567-72.
Citation 1:
Koziczak-Holbro et al. IRAK-4 kinase activity-dependent and -independent regulation of lipopolysaccharide-inducible genes. Eur J Immunol. 2008;38(3):788-96.PMID: 18266302
Citation 2:
Hatao et al. Prolonged Toll-like receptor stimulation leads to down-regulation of IRAK-4 protein. J Leukoc Biol. 2004;76(4):904-8. PMID: 15258191
Citation 3:
Heinz et al. The death domain-containing protein Unc5CL is a novel MyD88-independent activator of the pro-inflammatory IRAK signaling cascade. Cell Death Differ. 2012;19(4):722-31. PMID: 22158417
Citation 4:
Koziczak-Holbro et al. IRAK-4 kinase activity is required for interleukin-1 (IL-1)receptor- and toll-like receptor 7-mediated signaling and gene expression. J Biol Chem. 2007;282(18):13552-60. PMID: 17337443

FOR RESEARCH USE ONLY

For additional information, visit ProSci’s Terms & Conditions Page.

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.

CATALOG NUMBER:

3125

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

Datasheet
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