Description
Neurite adhesion molecule L1 has been implicated in neuron-neuron and neuron-Schwann cell adhesion in vertebrates. L1-like molecules, found in mouse, rat, chicken, and human, promote axonal elongation and may also play a role in regeneration of axons after injury. Molecular cloning data suggest 87% amino acid identity between mouse and human L1 molecules. 5G3 antigen (Ag), originally defined by monoclonal antibody 5G3, is considered to be the human homologue of mouse L1. The 5G3 antibody was developed against a human neuroblastoma cell line to use as a probe for the elucidating the biological characteristics of neuroblastoma. 5G3 specifically recognizes a neuroblastoma target glycoprotein antigen of 215 kDa and its 200 kDa precursor. The 215 kDa molecule is expressed on the cell surface; whereas the 200 kDa precursor is shed from the cell surface. The 215 and 200 kDa species also differ in their posttranslational modification patterns. The 5G3 antibody has been used as a marker for neuroblastoma, and to purify 5G3 Ag from normal adult human brain. The antibody recognizes human L1 on human neuroblastoma cell lines and tissues. Reactivity has been tested on a variety of malignant and normal tissues. Squamous lung, squamous skin, and osteogenic sarcoma cell lines were positive, as were two out of eight melanoma cell lines tested. A variety of other cell lines and tumor tissues tested negative. 5G3 did not react with either T or B lymphoblastoid cell lines or a fibroblast cell line. Among all the normal tissues tested, mAb 5G3 reacted only with cerebellum. The molecular masses observed using mAb 5G3 may vary among immunoprecipitation isolates. In normal human cerebellum, 5G3 Ag migrated as a 190/200 kDa doublet, 140 kDa band with minor bands at 80 and 65 kDa. 5G3 Ag isolated from SK-N-AS cells migrates as 200 to 215 kDa bands, or as a diffuse band ranging from 200 to 215 kDa. Additional bands have been described at 140 to 150 kDa in SK-N-AS cells. Only the 200 kDa band has been detected in culture media from SK-N-AS cells.
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions that minimize unconjugated dye and antibody.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
3. For U.S. patents that may apply, see bd.com/patents.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
6. The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
7. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
8. When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
9. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
10. An isotype control should be used at the same concentration as the antibody of interest.
11. Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
