Donkey Conjugate Line In many cases double labeling experiments are based on using two primary antibodies from different animal species, for instance mouse and rabbit. Of late, we have noticed an increasing number of users that have primary antibodies developed in goat. It is impossible to perform a double labeling experiment in combination with, for instance a mouse primary antibody, since applicable secondary antibodies are either raised in rabbit or goat. Hence the Goat-anti-Mouse would be recognized by the Rabbit-anti-Goat, and it would be impossible to get reliable results. In the past, only using directly labeled primary antibodies would solve this. This would require researchers to go through the lengthy procedure of preparing such conjugates and even then with limited success. To overcome this difficulty we are proud to release the Aurion Series of Secondary ImmunoGold conjugates based on antibodies raised in Donkey, including Donkey-anti-Goat. It is then known that mixing Donkey-anti-Goat and Donkey-anti-Mouse provides a successful solution to the above mentioned case. Ultra Small Immuno Gold Reagents Aurion Ultra Small Immunogold Reagents are prepared with subnanometer gold particles. These particles have far less influence on the adsorbed antibodies or detecting molecules, and consequently the conjugates behave as though they are uncoupled. In conjunction with the highly efficient and easy-to-use R-Gent SE-LM and SE-EM silver enhancement reagents, the Ultra Small Immunogold Reagents are the best choice for any application. Introduction Reduction of the gold particle size provides Ultra Small Immunogold Reagents with fundamentally different characteristics when compared with conjugates built around larger particles. While the Conventional Immunogold Reagents can be thought of as particles coated with proteins, Ultra Small Immunogold Reagents are proteins coated with one or more gold particles. With this structure, both the overall size of the conjugates, as well as steric hindrance are decreased. The table below lists a few physical characteristics of gold conjugates.
|Particle diameter||#Au atoms||MWt. (daltons)||#particles /ml||#Ab /(part.)|
The small gold particles also have a tight particle surface curvature which makes it less likely that a structured water dipole layer will build-up around the gold particles. Hence, the hydrodynamic radius of the ultra small gold colloids is reduced. Finally, small gold particles carry less net negative charge; thus, they undergo less charge determined repulsion when approaching the sample surface. Aurion offers Ultra Small Immunogold Reagents with an average gold particle diameter of 0.8 nm or less. These ultra small gold particles can be visualized directly in high angle annular dark-field-scanning TEM. However, the gold signal is normally visualized after increasing the particle diameter with silver enhancement. The reagents can be used in electron and light microscopy as well as in blotting experiments. The universal applicability makes it easy to compare results obtained with different procedures. Product Description AURION Ultra Small Immunogold Reagents contain 60-80 µg of specific protein/ml for IgG conjugates. F(ab’)2, Fab and biotinylated albumin conjugates contain equimolar amounts of conjugated protein. The average gold cluster diameter is less than 0.8 nm. AURION Ultra Small Immunogold Reagents are used in conjunction with AURION R-Gent SE-EM or SE-LM silver enhancement reagents, developed for electron microscopy and light microscopy/immunoblotting respectively. The reagents are supplied in PBS with 1% Bovine Serum Albumin and 15 mM NaN3. The activity of each lot is determined using a dot-spot test system as described by Moeremans et al., J. Immunol. Methods, 74, (1984), 353. The products are available in two package sizes: Regular package: for 1200 grids or 600 slides Small package: for 480 grids or 240 slides. Application Instructions Detailed information is provided in the package inserts Electron Microscopy Post-embedding Labeling Incubate thin sections by placing grids on drops of reagent arrayed on a sheet of Parafilm™. The use of nickel grids is recommended, especially if silver enhancement procedures are intended. Pre-embedding Labeling Float specimens in dilute reagent on a rocking table. There is a special word on pre-embedding in the “sharing our knowledge” section of this website as well as a Newsletter (free copy on request). The incubation protocol on pre-embedding can be found in the section “the Aurion method” Light Microscopy Slide or Coverslip Labeling Apply a few drops of dilute reagent to cover the specimen. Recommended Incubation Solution: PBS, (10 mM Phosphate buffer, 150 mM NaCl) 0.1-0.2 % AURION BSA-c™ 15 mM NaN3 pH 7.4 Note on background prevention: A special AURION Newsletter dealing with the topic of background is available on request. A special AURION Newsletter describes the use of AURION Ultra Small Immunogold Reagents in pre-embedding labeling. A free copy is available on request. Specificity Aurion offers the widest range of Ultra Small Immunogold Reagents. The most commonly used reagent types are available as intact IgG, F(ab’)2 and Single Fab conjugates. Please refer to ‘Ordering Information’ for a complete list of reagents available. AURION Ultra Small Immunogold Reagents are prepared using the highest quality antibodies or binding agents available. All antibodies are immuno affinity purified and immuno cross-adsorbed to reduce non-specific interactions. Storage AURION Immunogold Reagents have a guaranteed shelf life of 18 months from the date of quality control analysis. The products should be stored at 4-8°C. Freezing is not recommended. Conventional Immuno Gold Reagents Conventional Immunogold Reagents are available in four size classes. The monodisperse size population makes the conjugates suited for multiple labeling with no overlap. The Conventional Immunogold Reagents are the classical conjugates in immuno electron microscopy; they are a good choice when the antigen is abundant and the accessibility of the antigen is relatively good. Introduction The conventional labeling approach in transmission and scanning electron microscopy utilizes secondary immunogold reagents based on particles that can be observed without enhancement. These conjugates are suited for single and multiple labeling in electron microscopy, when the number of antigens available for binding is such that a relevant signal can be obtained. The AURION Conventional Immunogold Reagents are built around colloidal gold particles with sizes of 6, 10, 15 or 25 nm. The particle population is monodisperse and thus shows minimal size variation and overlap. Typically, the coefficient of variance for the 6 and 25 nm particle size conjugates is less than 12%, whereas the 10 and 15 nm size conjugates show less then 10% variation. The table below lists a few physical characteristics of gold conjugates:
|Particle diameter||#Au atoms||MWt. (daltons)||#particles /ml||#Ab /(part.)|
Product Description AURION Conventional Immunogold Reagents are tailored to contain 10-20 µg of specific protein/ml. The reagents are supplied in PBS with 1% Bovine Serum Albumin and 15 mM NaN3 at an OD520nm of 1.0 to warrant prolonged cluster free storage. The activity of each lot is determined using a dot-spot test system as described by Moeremans et al., J. Immunol. Methods, 74, (1984), 353. Actual lot specifications (size, variation and expiry date) are indicated on the accompanying package insert. Regular package: for the labeling of 1000-2000 grids Small package: for the labeling of 400-800 grids Application Instructions On-Grid Labeling For most applications grids are floated on top of drops of dilute reagent displayed on a sheet of Parafilm™. The use of gold or nickel grids is recommended. Whole mount and intact cell labeling Specimens are kept floating in dilute reagent on a rocking table. Recommended Incubation Solution: PBS, (10 mM Phosphate buffer, 150 mM NaCl), 0.1-0.2 % AURION BSA-C™ 15 mM NaN3 pH 7.4 Specificity Aurion offers the widest range of Conventional Immunogold Reagents. Please refer to the list of available reagents under ‘Ordering Information’. AURION Conventional Immunogold Reagents are prepared using the highest quality antibodies or binding agents available. All antibodies are immuno affinity purified and immuno cross-adsorbed to reduce non-specific reactions. Storage AURION Conventional Immunogold Reagents have a guaranteed shelf life of 18 months from the date of quality control analysis. The products should be stored at 4-8°C. Freezing is not recommended.