Antibody Insider—Issue 2017 Q4
Conjugated organelle-specific antibodies provide an easy-to-use tool for examining cellular structure
Antibodies to proteins found within specific organelles have been a useful tool in immunofluorescent microscopy to highlight structural morphology and sub-cellular localization. Identification of protein localization may provide clues to the role a particular protein may have within the cell. Organelle-specific antibodies are used to show co-localization of a target of interest and a known organelle specific protein, or to examine the location of fluorescently tagged protein constructs. Many of these organelle-specific antibodies are now available conjugated to Alexa Fluor dyes for easy use in immunofluorescence experiments without requiring the use of secondary antibodies.
Quickly visualize morphological features
As tools for looking at cell morphology, these Alexa Fluor dye–conjugated organelle antibodies can provide a quick and easy way to understand the structure of a particular cell type. In the figure below, an Alexa Fluor 488 anti-ZO-1 conjugate was used to highlight cell junctions formed during the culture of Caco-2 cells. Caco-2 cells can be cultured to become differentiated so that they look morphologically similar to enterocytes in the small intestine. The proper formation of ZO-1 detectable cell junctions is an indicator that the differentiation process was successful.
Multiplexed staining of cell junctions, mitochondria and nucleolus in Caco-2 cells. Caco-2 cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with 0.1% Triton™ X-100 in TBS for 15 minutes, and blocked with 3% Blocker BSA in PBS for 30 min at room temperature. Cells were stained with a ZO-1 monoclonal antibody, Alexa Fluor 647 conjugate (red) at 10 µg/mL, ATP Synthase beta mouse monoclonal antibody, Alexa Fluor 488 conjugate (green) at 5 µg/mL and Nucleophosmin monoclonal antibody, Alexa Fluor 488 conjugate (green) at 2.5 µg/mL in blocking buffer for 1 hr at room temperature protected from light. Nuclei (blue) were stained with Hoechst dye at a dilution of 1:10,000 in blocking buffer. Images were taken on an EVOS FL Instrument at 60x magnification.
These conjugated organelle-specific antibodies join a large selection of organelle specific dyes already available from Thermo Fisher Scientific, and can be used where antibody based approaches may provide an advantage. However, depending upon the experimental design, you can combine with dyes, or traditional primary plus secondary antibody detection.
Organelle antibodies offer broad utility
The conjugated organelle specific antibodies can be used in any application where a pre-conjugated antibody would simplify the experiment. For example instead of GAPDH as a loading control, you could use the signal from actin or tubulin for signal normalization. Alternatively, you could use organelle-specific antibodies in cell fractionation studies, potentially as a control showing that the organelles appeared in the expected cell fraction.
We currently offer over 30 organelle-specific antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647 dyes. Occasionally, we provide more than one potential marker as there may be some sample types or cell treatments where a particular canonical organelle marker is not expressed in the organelle as expected. However, in general these protein targets have been selected due to their known usefulness in examining organelle localization and fractionation.
Find more information on organelle antibodies and search for the organelle antibody you need at thermofisher.com/organelleabs
Quantify neuro-related proteins using quality Invitrogen ELISA kits
For scientists who study neurodegeneration, Alzheimer’s, Parkinson’s and other neuro-related conditions, enzyme-linked immunosorbent assays (ELISAs) are a simple and unbiased way to measure neuro-related proteins such as β-amyloid, α-Synuclein or tau from cerebral spinal fluid (CSF), brain homogenate, or tissue culture supernatant samples. Our Invitrogen Neuro-ELISA ready-to-use kits come in human and mouse species and include all the necessary reagents, including calibrated standards to allow for quantitative results. All kits are rigorously tested to help ensure accuracy and reproducibility.
- Sensitive—use small sample volume to detect proteins
- Consistent—achieve reproducible, quantitative results
- Fast—results in ~4 hours
Our assays are easy to run — Here’s how they work
The 96-well plates are pre-coated with an immobilized monoclonal antibody. The plates come in strip-wells, so you can choose to run as many or as few strips at a time. The sample and detection antibody are added, and the immobilized antibody binds to the protein antigen. The detector antibody binds to a different site on the protein. Nonspecific protein and unbound detector antibody are washed away. HRP-conjugated anti-rabbit antibodies are added and bind to the detector antibody. Unbound HRP-conjugated antibodies are washed away. A stabilized chromogen (substrate) is added to react with any bound HRP enzyme to produce a color, which is directly proportional to the concentration of the protein present. A stop solution is finally added to terminate the color reaction, and the color intensity (yellow) is measured using a plate reader.
The optical density of each dilution was plotted against the standard curve. Parallelism was demonstrated by the figure below and indicated that the standard accurately reflects Tau content in samples.
See the current list of neuro-related ELISAs at thermofisher.com/neuroELISA
Two new ABfinity antibodies now available for the study of neuronal synaptic vesicle exocytosis
SNARE proteins are important in the release of neurotransmitters through synaptic vesicle fusion and exocytosis. Synaptosomal-associated protein of 25 kDa (SNAP-25) and vesicle associated membrane protein-1 (VAMP1/Synaptobrevin1/Syb) are two major SNARE proteins that mediate synaptic vesicle exocytosis. A dominant negative mutation in SNAP-25 leads to impaired vesicle trafficking in a mouse model, and other mutations are a novel cause of epilepsy and genetic disability. VAMP1 mutations have been reported in congenital myasthenic syndrome and hereditary spastic ataxia.
VAMP1 and synaptic protein, Syntaxin, interact with SNAP25 after the latter forms a binary complex with another protein, tagmin. This interaction plays a critical role in synaptic vesicle fusion to the presynaptic membrane.
In mouse hippocampal neurons, the ABfinity anti SNAP-25 antibody detects SNAP-25 that is enriched in the neuronal processes (see figure).
Expression of SNAP-25 in primary mouse hippocampal neurons by immunofluorescence using ABfinity SNAP-25 monoclonal antibody.
In western blotting, the ABfinity anti SNAP-25 antibody detected a single band at the expected molecular weight in PC12 cell line (see figure). Reduced expression of SNAP-25 was detected in SNAP-25 siRNA transfected lysates compared to un-transfected and scrambled controls, confirming that the observed band indeed corresponds to
The Invitrogen ABfinity anti VAMP-1 antibody detects a single band by western blot corresponding to the expected molecular weight of VAMP1 in PC12 cells, rat, and mouse brain tissue. As expected, VAMP1 is detected in NGF - differentiated PC12 cells but not in undifferentiated cells (see figure).
Data support the high specificity claim of Invitrogen recombinant ABfinity anti SNAP-25 antibody and Invitrogen ABfinity anti VAMP-1 antibody to their respective targets, and their performance in applications that are most routinely used to study these proteins. These antibodies fit in the study of neuronal behavior during disease and development by enabling researchers to design and conduct experiments in biologically appropriate model systems.
To find these and other ABfinity antibodies go to thermofisher.com/antibodies
Read the application note on these two ABfinity antibodies.
Increase neuronal cell survival by more than 50%—the highest in the industry
New Gibco B-27 Plus Neuronal Culture System
Since 1993, Gibco B-27 Supplement and Neurobasal Medium have been a trusted standard for a variety of neuronal culture applications, with citations in more than 11,000 publications. However, as the desire for more reliable and biologically relevant models has increased, so too has the necessity for a media system that can maintain and mature optimal densities of functional neurons over longer periods of time in vitro. To meet this need, we have developed the new Gibco B-27 Plus Neuronal Culture System. It features an optimized formulation, upgraded manufacturing process, and more stringent quality control of raw materials and final product. These improvements enable:
- At least 50% higher survival of primary rat neurons compared to classic B-27 Supplement and Neurobasal Medium
- Highest survival of primary rat, mouse, and human stem cell-derived neurons compared to other commercially available serum-free neuronal media systems
- Improved electrophysiological activity and maturation—more consistent, stable, and synchronized spontaneous activity in primary rodent neurons after 10 days compared to BrainPhys™ Neuronal Medium
- Accelerated neurite outgrowth and increased the number of synaptic-positive puncta
B-27 Plus Neuronal Culture System maintains higher neuronal survival rates compared to B-27 Supplement and Neurobasal Medium. Cryopreserved Rat Cortex Neurons were cultured for up to 3 weeks in the listed media system and then immunostained with MAP2 on days 7, 14, and 21.
The Gibco B-27 Plus Neuronal Culture System includes
- B-27 Plus Supplement (50X)—10mL
- Neurobasal Plus Medium—500mL
Seamlessly replace other neuronal cell culture systems, such as classic B-27 and Neurobasal in maintenance/maturation and differentiation protocols with no change to your current workflow.
Try the B-27 Plus Neuronal Culture System in your experiments today. Get your free sample at thermofisher.com/b27plus
Using Brilliant Violet antibodies? Meet the Super Bright alternative
The eBioscience Super Bright antibody conjugates for flow cytometry are now available in an even greater variety of markers, clones and formats! With over 400+ products covering 100+ different clones (includes both human and mouse antigens), Super Bright antibody conjugates provide options in marker and clone selection when purchasing violet laser excitable antibody conjugates for flow cytometry.
Discriminate dim cell populations more easily using Super Bright 436, Super Bright 600, Super Bright 645 and Super Bright 702 antibody conjugates; these conjugates display similar or better brightness to Brilliant Violet™ conjugates and some have been shown to require less compensation. Discover your options and find a Super Bright antibody conjugate to fit your flow cytometry panel today.
Better resolution achieved with Super Bright antibody conjugates vs. APC antibody conjugates. Human peripheral blood mononuclear cells (PBMCs) were stained with PE anti–human CD20 antibody and anti–human CD11c antibody conjugated either to Super Bright 702 dye (left) or to APC (right). CD11c+ cells stained with Super Bright antibodies demonstrate better resolution than those stained with APC antibodies.
Thermo Fisher Scientific protein and cell analysis education resource
With this free virtual learning platform, you can access webinars, posters, and papers about applications and techniques related to protein gel chemistries, western detection, mass spectrometry, flow cytometry, fluorescence imaging, high-content imaging, antibodies, immunoassays, and other topics.
Learn more, visit thermofisher.com/proteincelledu
Representative on-demand webinars
Flow with the experts
Do you use a flow cytometer? Ever wonder about the true definition of an event rate and if you should trust the “maximum event rate” of your flow cytometer? Visit the “Behind the Bench” blog by Thermo Fisher Scientific to learn directly from our Research and Development experts.
Introducing an easier way to find Invitrogen antibodies
Our new antibody shopping experience offers a streamlined interface and a simpler way to find the high quality Invitrogen antibodies you’re searching for. Go to thermofisher.com/antibodies to see the new look and improved experience.
Short video: Antibody validation through advanced specificity verification
Antibodies are some of the most critical reagents used in life science and medical research. However, the use of poorly characterized antibodies can generate significant problems–compromising research projects and costing valuable time and money.
Watch the short video to learn more about Thermo Fisher Scientific advanced verification standards, where Invitrogen antibodies are tested for both target specificity and robust application validation.*
Watch the fun and bright adventures of Alexa Fluor secondary antibody
For Research Use Only. Not for use in diagnostic procedures.