- Predicted Species Reactivity:
- Product Format:
- Purified IgG - liquid
- Additional Information:
- Fusion Partners: Spleen cells from immunised BALB/c mice were fused with cells of the mouse P3X63.Ag 8653 myeloma cell line.
- Preparation: Purified IgG prepared by affinity chromatography on Protein A
Preservative Stabilisers: 0.09% Sodium Azide (NaN3)
0.1% Bovine Serum Albumin
- Approx Protein Conc: IgG concentration 1.0 mg/ml
Buffer Solutions: Phosphate buffered saline, pH 7.2
- Reconstitution and Storage:
- Store at +4oC or at -20oC if preferred.
This product should be stored undiluted.
Storage in frost-free freezers is not recommended. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
- NCBI Gene Id:
- Protein Name:
- CD40 ligand
- Alias Symbols:
- IGM, IMD3, TRAP, gp39, CD154, CD40L, HIGM1, T-BAM, TNFSF5, hCD40L, CD40LG
- Replacement Item:
- This antibody may replace item sc-1593 from Santa Cruz Biotechnology.
- Description of Target:
- MOUSE ANTI HUMAN CD154 (CD40L)
- Protein Size (# AA):
- Tissue Tool:
- Find tissues and cell lines supported by DNA array analysis to express CD40LG.
- RNA Seq:
- Find tissues and cell lines supported by RNA-seq analysis to express CD40LG.
- The immunogen for anti-CD40LG antibody: mouse myeloma cell line transfected with human CD40L (CD154)
- Predicted Homology Based on Immunogen Sequence:
- Printable datasheet for anti-CD40LG antibody - OASA01316
- Application Info:
- Flow Cytometry: 1/10 - 1/50
- Protocol Information:
- Citation: 1: Bugajska U, Georgopoulos NT, Southgate J, Johnson PW, Graber P, Gordon J,Selby PJ, Trejdosiewicz LK. The effects of malignant transformation onsusceptibility of human urothelial cells to CD40-mediated apoptosis. J NatlCancer Inst. 2002 Sep 18;94(18):1381-95. PubMed PMID: 12237284.
Experiment Name: 1. Flow Cytometric Quantitation of Cell Surface Antigen Expression2. Single- and double-label indirect immunofluorescence
Experiment Background: 1. The tumor necrosis factor (TNF) superfamily of ligands and receptors mediates immune cell survival. Some members possess a death domain, a protein motif that functions to transmit apoptotic signals, whereas others, such as CD40, do not. CD40 is expressed by both normal and malignant epithelial cells. To investigate the functional significance of this expression, we studied the effects of ligation of CD40, Fas, and TNF receptors (TNFRs) on the proliferation and survival of normal and malignant human urothelial cells and urothelial cells with disabled p53 function.2. Expression of CD40 on urothelial cells and CD40L on mitomycin C-treated 3T3CD40L and CD40L-L fibroblasts was determined by flow cytometry. 3. Indirect immunofluorescence microscopy revealed that NFκB was localized to the cytoplasm in both normal and malignant urothelial cells; incubation of those cells with sCD40L, TNF-α, or anti-CD95 antibody did not induce the nuclear translocation of NFκB.
Experimental Steps: 1. Flow Cytometric Quantitation of Cell Surface Antigen Expression: E xpression of CD40 and CD40L was determined by incubation of cells with unconjugated specific primary antibodies (Oxford, U.K.), followed by phycoerythrin-conjugated goat anti-mouse Ig (F[ab]’2 fragment) (Southern Biotechnology Associates, supplied by Eurogenetics, Hampton, U.K.). All antibodies were titrated before use and diluted in 0.2 µm-filtered phosphate-buffered saline (PBS) containing 1% FBS and 0.1% NaN3. Cells were harvested, and antibody incubations were carried out. Cells were analyzed on a FACScan flow cytometer using CellQuest software (BD Biosciences). At least 3000 events (i.e., cells) were acquired from each sample. The baseline median fluorescence channel was established for each cell line with the use of control cells that were incubated with either an irrelevant antibody to glucose oxidase from Aspergillus niger (Dako Ltd., High Wycombe, U.K.) or no antibody.2. Single- and double-label indirect immunofluorescence: NFκB was localized by using a rabbit polyclonal antibody (NFκB p65; Santa Cruz, Insight Biotechnology, London, U.K.). Urszula et al. used a mouse monoclonal antibody to CK8 (Zymed) to identify urothelial cells and an anti-CD40L mouse monoclonal antibody to identify 3T3CD40L fibroblasts. All antibodies were used at pretitrated optimal dilutions. Goat anti-rabbit Ig–Texas Red and anti-mouse–FITC conjugates (Southern Biotechnology Associates) were used to visualize the immunolabeling patterns. As positive controls for NFκB translocation to the nucleus, human foreskin-derived fibroblasts were treated with TNF-α and processed for immunofluorescence localization of NFκB.
Number Of Protocols: 2
- Target Reference:
- 1. Hermann, P. et al. (1993) Expression of a 32-kDa ligand for the CD40 antigen on activated human T lymphocytes. Eur. J. Immunol. 23: 961-964.
2. Lane, P. et al. (1992) Activated human T cells express a ligand for the human B cell associated antigen CD40 which participates in T cell-dependent activation of B lymphocytes. Eur. J. Immunol. 22: 2573-2578.
3. Kroczek, R.A. et al. (1994) Defective expression of CD40 ligand on T cells causes "X-linked immunodeficiency with hyper-IgM (HIGM1)" Immunol. Rev. 138: 39-59.