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<strong>Figure 1 Western Blot Validation with Recombinant Protein</strong><br>Loading: 30 ng of human MD-2 recombinant protein per lane.Antibodies: MD-2, 3289 (Lane 1: 0.5 μg/mL, Lane 2: 1 μg/mL and Lane 3: 2 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 Western Blot Validation in Daudi Cell Lysate</strong><br>Loading: 15 μg of lysates per lane.Antibodies: MD-2, 3289 (1 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 Mouse Spleen Tissue Lysate</strong><br>Loading: 15 μg of lysates per lane.Antibodies: MD-2, 3289 (Lane 1: 0.5 μg/mL and Lane 2: 1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Western Blot Validation in Mouse Tissue Lysates</strong><br>Loading: 15 μg of lysates per lane.Antibodies: MD-2, 3289 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Western Blot Validation in Rat Spleen Tissue Lysate</strong><br>Loading: 15 μg of lysates per lane.Antibodies: MD-2, 3289 (Lane 1: 0.5 μg/mL and Lane 2: 1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 6 Western Blot Validation in Rat Tissue Lysates</strong><br>Loading: 15 μg of lysates per lane.Antibodies: MD-2, 3289 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 7 Immunohistochemistry Validation of MD-2 in Human Spleen Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded human spleen tissue using anti-MD-2 antibody (3289) at 2.5 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 MD-2 in Human Spleen Tissue</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed human spleen tissue labeling MD-2 with 3289 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
<strong>Figure 9 Immunohistochemistry Validation of MD-2 in Rat Spleen Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded rat spleen using anti-MD-2 antibody (3289) 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 10 Immunofluorescence Validation of MD-2 in Rat Spleen Tissue</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed rat spleen tissue labeling MD-2 with 3289 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
Figure 1 Western Blot Validation with Recombinant Protein
Loading: 30 ng of human MD-2 recombinant protein per lane.Antibodies: MD-2, 3289 (Lane 1: 0.5 μg/mL, Lane 2: 1 μg/mL and Lane 3: 2 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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MD-2 Antibody

CATALOG NUMBER: 3289

Clonality:
Polyclonal
Tested Applications:
ELISA, IF, IHC-P, WB
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Conjugate:
Unconjugated
Specifications
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Immunogen:
Anti-MD-2 antibody (3289) was raised against a peptide corresponding to 13 amino acids near the center of human MD-2.
The immunogen is located within the last 50 amino acids of MD-2.
Conjugate:
Unconjugated
Tested Applications:
ELISA, IF, IHC-P, WB
Application Note:
WB: 0.5-1 μg/mL; IHC: 2-2.5 μg/mL; IF: 10-20 μg/mL.
Antibody validated: Western Blot in human, mouse and rat samples; Immunohistochemistry in human and rat samples; Immunofluorescence in human and rat samples. All other applications and species not yet tested.
Positive Control 1:
Cat. No. 1406 - Mouse Spleen Tissue Lysate
Positive Control 2:
Cat. No. 1466 - Rat Spleen Tissue Lysate
Positive Control 3:
Cat. No. 10-901 - Human Spleen Tissue Slide
Predicted Molecular Weight:
Predicted: 19kD
Observed: 26 kD (Post-modification: 2 N-linked glycosylations)
Validation:

Recombinant Protein Test (Figure 1): Anti-MD-2 antibodies (3289) detected human MD-2 recombinant protein at different concentrations.

Isoforms:
Human MD-2 has 2 isoforms, including isoform 1 (160aa, 18kD) and isoform 2 (130aa, 15kD). Mouse MD-2 has one isoform (160aa, 18kD) and Rat MD-2 also has one isoform (160aa, 18kD). 3289 can detect human, mouse and rat.
Purification:
MD-2 Antibody is affinity chromatography purified via peptide column.
Clonality:
Polyclonal
Isotype:
IgG
physical-state:
Liquid
Buffer:
MD-2 Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Storage Conditions:
MD-2 antibody can be stored at 4°C up to one year. Antibodies should not be exposed to prolonged high temperatures.
Ncbi Official Symbol:
LY96
Additional Names:
MD-2 Antibody: MD2, MD-2, ly-96, ESOP-1, ESOP1, MD2, Lymphocyte antigen 96, Ly-96
Protein Accession Number:
NP_056179
Protein Gi Number:
223555998
Ncbi Gene Id Number:
23643
User Note:
Optimal dilutions for each application to be determined by the researcher.
Background:
MD-2 Antibody: MD-2 is a member of the Toll/interleukin-1 receptor (TIR) family, a group of proteins that include the Toll-like receptors (TLRs). TLRs are signaling molecules that recognize different pathogen-associated molecular patterns (PAMPs) and serve as an important link between the innate and adaptive immune responses. TLR4, the major signaling receptor for lipopolysaccharide (LPS), requires the binding of MD-2 to its extracellular region for maximal response to LPS. The specificity of this response is determined by the species of MD-2; e.g., human MD-2 can cause mouse TLR4 to react to LPS analogs that are normally antagonistic to human but not mouse TLR4.
Background Reference 1:
O'Neill et al. Trends in Imm. 2003; 24:286-9.
Background Reference 2:
Vogel. Mol. Interv. 2003; 3:466-77.
Background Reference 3:
Takeda et al. Annu. Rev. Immunol. 2003; 21:335-76.
Background Reference 4:
Janeway et al. Annu. Rev. Immunol. 2002; 20:197-216.

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:

3289

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

Datasheet
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