货号: | 7255 |
数量: | 99 |
保质期: | 1-2年 |
保存条件: | 4℃ |
英文名: | FluoroNanogold™-anti-guinea pig Fab'-Alexa Fluor®* 488 |
供应商: | Nanoprobes |
规格: | 0.5 mL |
Product Information
Alexa Fluor®FluoroNanogoldTMis a unique, dual-purpose probe. Alexa Fluor®488 FluoroNanogoldTMreagents consist of affinity-purified Fab' fragments conjugated to both Alexa Fluor®488 dye (green) and the 1.4 nm Nanogold®particle.1An Alexa Fluor®488 FluoroNanogoldTMFab conjugate is shown in Figure 1. In the fluorescence microscope, these probes may be used just like conventional fluorescently-labeled antibodies,2while in the electron microscope they are visualized in exactly the same manner as for Nanogold®conjugates.3The combination of the fluorescent and electron microscopy, or referred as correlative microscopy,13allows imaging of the same exact structure in both microscopes. The covalent label linkage is stable indefinitely, and the attachment at a hinge thiol site ensures maximum preservation of native immunoreactivity. These reagents are supplied at a concentration of 0.08 mg/mL of Fab' dissolved in 20 mM phosphate buffered saline (150 mM NaCl) at pH 7.4 (PBS), with 0.1% BSA and 0.05% sodium azide as preservatives. The degree of labeling for each conjugate is typically one Nanogold®particle and 2-3 fluorophores per Fab molecule, and the exact degree of labeling is indicated on the product specification sheet. FluoroNanogoldTMconjugates should be stored at 2-8°C. DO NOT FREEZE. PROTECT FROM LIGHT.
Figure 1: Fab' covalently conjugated to Alexa Fluor® 488 and NANOGOLD® to give Alexa Fluor® FluoroNanogoldTM.
General Considerations for Immunostaining with FluoroNanogoldTMReagents
Basically, normal methodologies for each component of the label may be used successfully with Alexa Fluor®FluoroNanogoldTMlabeling agents. Due to some quenching of fluorescence by the gold particle, slightly higher concentrations of antibody are recommended for incubations. A blocking agent of 5% non-fat dried milk has been found to reduce nonspecific background staining in some cases: this should be used before incubation with probe (in standard wash/blocking steps), and additionally, the Alexa Fluor®FluoroNanogoldTMprobe can be diluted in a solution also containing 1% non-fat dried milk before it is applied.
Properties
- Alexa Fluor®FluoroNanogoldTMcontains an extremely uniform 1.4 nm diameter gold particle (±10%).
- The absorption maximum of Alexa Fluor®488 occurs near 494 nm, and the emission maximum is near 519 nm. These values are very similar to those of fluorescein (green), so a fluorescein filter set is recommended for fluorescence observation.
- Alexa Fluor®FluoroNanogoldTM-Fab is smaller than a single whole IgG molecule. It is not significantly larger than Fab'- NanogoldTM, the smallest gold immunoprobe commercially available, and will penetrate and reach antigens inaccessible to other gold probes.
- Alexa Fluor®FluoroNanogoldTM-Fab is chromatographically purified through gel filtration columns. There are absolutely no aggregates or other molecular weight impurities. This is in sharp contrast to colloidal gold conjugates which usually are prepared by centrifugation to remove the largest aggregates, and frequently contain smaller aggregates.
- Close to 1 NanogoldTMlabel to 1 Fab' make this product distinct from the 0.2 - 10 variable stoichiometry of colloidal gold - antibody preparations.
- Alexa Fluor®FluoroNanogoldTMconjugates are usually used as secondary antibody. They may also be used as a tertiary labeled antibody, or a custom Alexa Fluor®FluoroNanogold TMconjugate may be the primary antibody.
- Alexa Fluor®FluoroNanogoldTMparticles do not have affinity to proteins as do colloidal golds. This reduces background and false labeling.
- Alexa Fluor®FluoroNanogoldTMdevelops better with silver than do most colloidal golds giving it higher sensitivity. Both silver and gold enhancement can be used to make the immunolabeling useful for electron microscopy and light microscopy.
Using EM Stains with Alexa Fluor®FluoroNanogoldTM
Because the 1.4 nm Alexa Fluor®FluoroNanogoldTMparticles are so small, over staining with OsO4, uranyl acetate or lead citrate may tend to obscure direct visualization of individual Nanogold®particles. Four recommendations for improved visibility of Alexa Fluor®FluoroNanogoldTMare:
- Use of reduced amounts or concentrations of usual stains.
- Use of lower atomic number stains such as NanoVanTM, a Vanadium based stain.6
- Enhancement of Alexa Fluor®FluoroNanogoldTMwith silver developers, such as LI SilverTMor HQ SilverTM.
- Enhancement of Alexa Fluor®FluoroNanogoldTMwith the gold developer, GoldEnhanceTM.