|Product Name||Anti-CD40L/CD40LG Antibody|
|Storage & Handling||At -20°C for one year. After reconstitution, at 4°C for one month. It can also be aliquotted and stored frozen at -20°C for a longer time.Avoid repeated freezing and thawing.|
|Description||Rabbit IgG polyclonal antibody for CD40 ligand(CD40LG) detection. Tested with WB, IHC-P in Human.|
|Cite This Product||Anti-CD40L/CD40LG Antibody (Boster Biological Technology, Pleasanton CA, USA, Catalog # PA1020)|
|Immunogen||A synthetic peptide corresponding to a sequence at the N-terminus of human CD40L(47-65aa HRRLDKIEDERNLHEDFVF), different from the related mouse sequence by three amino acids, rat sequence by four amino acids.|
Assay Dilutions Overview
Western blot, 0.1-0.5μg/ml, Human
Boster's Secondary Antibodies And IHC, WB Kits
The following reagents are used to generate the images below.Boster recommends Enhanced Chemiluminescent Kit with anti-Rabbit IgG (EK1002) for Western blot, and HRP Conjugated anti-Rabbit IgG Super Vision Assay Kit (SV0002-1) for IHC(P).
Images And Assay Conditions
Figure 1. Western blot analysis of sCD40L using anti- sCD40L antibody (PA1020).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: MCF-7 Whole Cell Lysate,
Lane 2: HELA Whole Cell Lysate,
Lane 3: JURKAT Whole Cell Lysate,
Lane 4: HMY2 Whole Cell Lysate,
Lane 5: COLO320 Whole Cell Lysate.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti- sCD40L antigen affinity purified polyclonal antibody (Catalog # PA1020) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for sCD40L at approximately 29KD. The expected band size for sCD40L is at 29KD.
Figure 2. IHC analysis of sCD40L using anti- sCD40L antibody (PA1020).
sCD40L was detected in paraffin-embedded section of rat intestine tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti- sCD40L Antibody (PA1020) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.
Figure 3. IHC analysis of sCD40L using anti- sCD40L antibody (PA1020).
sCD40L was detected in paraffin-embedded section of human mammary cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti- sCD40L Antibody (PA1020) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.
Protein Target Info (Source: Uniprot.org)
|Protein Name||CD40 ligand|
|Tissue Specificity||Specifically expressed on activated CD4+ T- lymphocytes.|
|Alternative Names||CD40 ligand;CD40-L;T-cell antigen Gp39;TNF-related activation protein;TRAP;Tumor necrosis factor ligand superfamily member 5;CD154;CD40 ligand, membrane form;CD40 ligand, soluble form;CD40LG;CD40L, TNFSF5, TRAP;|
|Subcellular Localization||Cell membrane; Single-pass type II membrane protein.|
|Molecular Weight||29274 MW|
*if product is indicated to react with multiple species, protein info is based on the human gene.
|Protein Function||Mediates B-cell proliferation in the absence of co- stimulus as well as IgE production in the presence of IL-4. Involved in immunoglobulin class switching. .|
|Background||CD40 ligand(CD40L) is a type II membrane protein of 261 amino acids on activated T cells that induces B cell proliferation and immunoglobulin secretion. It has homology with tumour necrosis factor-alpha and -beta, and has important functions in B-cell activation and differentiation. Human CD40L with 5 exons, is mapped to the proximal region of the mouse X chromosome on Xq26.3-27.1, and can be detected on T cells but is absent from B cells and monocytes. Since CD40L is expressed on platelets and released from them on activation, its predictive value as a marker for clinical outcome and the therapeutic effect of inhibition of glycoprotein IIb /IIIa receptor in patients with acute coronary syndromes was investigated. The soluble CD40L may be involved in the process of restenosis and that it exerts its effect by triggering a complex group of inflammatory reactions on endothelial and mononuclear cells. CD40L plays a central role in the pathophysiology of acute coronary syndromes, and has a role in the pathogenesis of coronary artery lesions.|
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1. Post-translational modification:phosphorylation, methylation, glycosylation etc. These modifications prevent SDS molecules from binding to the target protein and thus make the band size appear larger than expected
2. Post-translational cleavage: this can cause smaller bands and or multiple bands
3. Alternative splicing: the same gene can have alternative splicing patterns generating different size proteins, all with reactivities to the antibody.
4. Amino Acid R chain charge: SDS binds to positive charges. The different size and charge of the Amino Acid side chains can affect the amount of SDS binding and thus affect the observed band size.
5. Multimers: Multimers are usually broken up in reducing conditions. However if the interactions between the multimers are strong, the band may appear higher.,