APEX Antibody Labeling Kits

what they are
APEX Antibody Labeling Kits provide a convenient means to directly attach a fluorophore to very small amounts of IgG antibody (10–20 µg).

how they work
These kits use a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX antibody labeling tip. This enables you to covalently label antibodies that are supplied in solutions containing stabilizing proteins, such as BSA or other contaminants, that can interfere with the amine-reactive labeling reagents used to attach the fluorophore to the antibody. Any contaminants are simply eluted through the tip. After the amine-reactive label is applied, a fluorescent IgG conjugate is ready for use in an imaging or flow cytometry application in as little as 2.5 hr with very little hands-on time.

what they offer

  • labeled antibodies ready for use in 2.5 hr (~15 min hands-on time)
  • covalent attachment of the label
  • labeling compatible with stabilizing proteins such as BSA

 

 Mitochondrial and Golgi complex labeling in HeLa cells. Fixed and permeabilized HeLa cells were treated with multiple mouse primary antibodies directly conjugated with APEX Alexa Fluor® Antibody Labeling Kits. The Golgi apparatus was detected with an anti–golgin-97 mouse monoclonal antibody labeled using the APEX Alexa Fluor® 555 Antibody Labeling Kit (Cat. no. A10470, orange fluorescence). Mitochondria were detected with an anti–OxPhos Complex V inhibitor protein mouse IgG1 monoclonal antibody labeled using the APEX Alexa Fluor® 488 Antibody Labeling Kit (Cat. no. A10468, green fluorescence). Nuclei were stained with blue-fluorescent DAPI (Cat. no. D1306, D3571, D21490).

 

Products Quantity Cat. no.
APEX Alexa Fluor® 488 Antibody Labeling Kit1 kitA10468
APEX Alexa Fluor® 555 Antibody Labeling Kit1 kitA10470
APEX Alexa Fluor® 594 Antibody Labeling Kit1 kitA10474
APEX Alexa Fluor® 647 Antibody Labeling Kit1 kitA10475
APEX Oregon Green® 488 Antibody Labeling Kit1 kitA10476
APEX Pacific Blue™ Antibody Labeling Kit1 kitA10478

 

The Countess™ Automated Cell Counter

what it is
The Countess™ Automated Cell Counter brings automation into the cell culture lab, counting cell samples in just 30 seconds. The instrument also provides a percent viability calculation and cell size distribution data and includes a handy dilution calculator and data archiving functions.

how it works
After staining with trypan blue is completed, 10 µl of a cell sample is placed in a highly accurate chamber slide, which is then inserted into the instrument. The Countess™ instrument then uses a sophisticated image capture and analysis technology to count the number of live and dead cells in the sample and measure the size of each cell.

what it offers

  • high accuracy and minimal subjectivity in cell counts and viability measurements
  • improved downstream results from better counting
  • possibilities for a wider range of experimental designs

 

 The Countess™ Automated Cell Counter. The kit includes the Countess™ instrument, one box of 50 chamber slides, 2 ml of trypan blue stain, and a USB drive.

 

Products Quantity Cat. no.
Countess™ automated cell counter1 kitC10227
Countess™ cell counting chamber slides *100 countsC10228
* Includes one box of 50 chamber slides and 2 ml of trypan blue stain.



For more information, visit www.invitrogen.com/countess.

Fluorescent and reactive azides and alkynes

what they are

The click chemistry product toolbox now includes azide- and alkyne-containing reagents for use in click chemistry reactions, including several of the best Alexa Fluor® azides for flow and imaging applications, and a larger unit size EdU for in vivo applications for detection of nascent DNA (S-phase) synthesis.

how they work

Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moieties to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Unlike traditional chemical reactions using succinimidyl esters or maleimides that target amines and sulfhydryls—functional groups that are not unique—click chemistry–labeled molecules can be applied to complex biological samples and detected with unprecedented sensitivity, thanks to extremely low background.

what they offer

  • the reaction between the detection moieties is efficient
  • no extreme temperatures or solvents are required
  • the reaction product is stable
  • the components of the reaction are bioinert
  • no side reactions occur—the label and detection tags react selectively and specifically with one another

 

Relative size of detection molecules commonly used in cellular analysis. Because the azide and alkyne moieties can be used interchangeably to optimize labeling configurations, R1 can be either the biomolecule of interest or the detection reagent. For biotin and the fluorophore, R2 is the biomolecule of interest.

Products Quantity Cat. no.
Alexa Fluor® 488 azide (Alexa Fluor® 488 5-carboxamido-(6-azidohexanyl), bis(triethylammonium salt)), 5-isomer0.5 mgA10266
Alexa Fluor® 647 azide, triethylammonium salt0.5 mg

A10277

Alexa Fluor® 594 azide (Alexa Fluor® 594 carboxamido-(6-azidohexanyl), triethylammonium salt), mixed isomers0.5 mgA10270
Oregon Green® 488 azide (Oregon Green® 6-carboxamido-(6-azidohexanyl), triethylammonium salt), 6-isomer0.5 mgO10180
alkyne, succinimidyl ester (3-propargyloxypropanoic acid, succinimidyl ester)1 mgA10279
azide, succinimidyl ester (azido polyethylene glycol (PEG4), succinimidyl ester)1 mgA10280
EdU (5-ethynyl-2'-deoxyuridine)500 mgE10187



Learn more about Click-iT™ products at www.invitrogen.com/click.

WesternDot™ 625 Western Blot Kits

what they are

The WesternDot™ 625 Western Blot Kits combine the bright fluorescence properties of Qdot® 625 nanocrystals with the high-affinity streptavidin–biotin interaction to allow simple, highly sensitive detection of proteins immobilized on nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes. Eliminate the use and cost of photographic film, darkrooms, or developing reagents.

how they work
Incorporating a standard western blotting protocol, the detection step relies on a biotinylated secondary antibody, either goat anti–rabbit IgG or goat anti–mouse IgG, followed by the key component, the Qdot® 625 streptavidin conjugate. The fluorescent signal is compatible with the commonly used modes of fluorescence detection for DNA or protein gels, and does not require specialized emission filters. Blots may be imaged wet or dry, and by epi- or transillumination.

what they offer

  • save time and hassle—no more frustrating ECL kit optimization
  • reduce costs—eliminate the need for X-ray films and developer
  • everything is included—ready to go out of the box

 

 

Comparison of WesternDot™ and ECL™ workflows. Both protocols have very similar steps and execution times. However, WesternDot™ detection is optimized right out of the box, whereas ECL™ blots (GE Healthcare) often require several rounds of detection to optimize film exposure and antibody concentrations.

 

 WesternDot™ 625 kits provide optimized detection right out of the box. (A) Typical WesternDot™ 625 detection result. (B) ECL™ detection (GE Healthcare) may require several different exposure runs to obtain the optimal result; as shown here, this may not be possible when wide ranges of protein concentration are present.

 

Products Quantity Cat. no.
WesternDot™ 625 Goat Anti-Mouse IgG Western Blot Kit20 minigel blotsW10132
WesternDot™ 625 Goat Anti-Rabbit IgG Western Blot Kit20 minigel blots

W10142


Learn more at www.invitrogen.com/westerndots.

Fluorescent lectin conjugates
Lectins are carbohydrate-binding proteins that bind to specific configurations of sugar molecules, such as oligosaccharides, present in cellular proteoglycans, glycoproteins, and glycolipids. Oligosaccharides are abundant on the cell surface and sometimes attached to certain constituents within the cell. Because the exposed carbohydrates are often unique to a given cell type or structure, lectins can be used to identify cell types or label cellular components in much the same way that an antibody or organelle stain might be used. This property makes them versatile detection reagents for microscopy and flow cytometry, and in gels. Lectins can also be used to mark cancer cells, as those cells often display altered surface glycoproteins. These conjugates offer all the benefits of the Alexa Fluor® dyes, including brightness, photostability, and color selection.

 
Staining with fluorescent lectin conjugates.
Capillaries in the hippocampal region of a mouse brain cryosection were visualized with the green-fluorescent Alexa Fluor® 488 conjugate of lectin HPA from Helix pomatia, which specifically binds to type A erythrocytes and α-N-acetylgalactosaminyl residues. Nuclei were stained with nuclear yellow.

 

Products Quantity Cat. no.
wheat germ agglutinin, Alexa Fluor® 488 conjugate5 mgW11261
concanavalin A, Alexa Fluor® 488 conjugate5 mgC11252
lectin HPA from Helix pomatia (edible snail), Alexa Fluor® 488 conjugate1 mgL11271


Learn more about our fluorescent lectin conjugates in our online Handbook of Fluorescent Probes and Research Products.

Interaction of a DNA intercalator DRAQ5, and a minor groove binder SYTO17, with chromatin in live cells—influence on chromatin organization and histone-DNA interactions.
Wojcik, K. and Dobrucki, J.W. (2008) Cytometry 73A:555–562.

Do DNA-binding dyes alter the structure of their target? Nucleic acid–binding dyes are widely used to provide information about the local concentration and distribution of DNA in the nuclei of living cells. However, dye binding may involve structural changes in chromatin that could alter our view of its organization in vivo. To investigate the impact of dye binding on chromatin structure, Wojcik and Dobrucki examined changes in the chromatin of HeLa cells expressing eGFP-tagged linker (H1) and core (H2B) histones. Binding of DRAQ5—a DNA intercalator—was accompanied by chromatin aggregation, even in the presence of as little as 1 µM dye. Increasing the dye concentration to 3 µM led to further aggregation and gradual loss of chromatin organization; 7.5 µM DRAQ5 was sufficient to randomize the chromatin distribution to the point where heterochromatin and euchromatin were no longer discernible. Fluorescence microscopy also revealed the detachment of H1 and (to a lesser degree) H2B histones as a result of DRAQ5 binding. The team then examined the effect of Molecular Probes® SYTO® 17 dye—a minor-groove binder—on chromatin structure. At SYTO® 17 dye concentrations of up to 7.5 µM, only slight aggregation of chromatin was observed; no histone detachment was detected over this concentration range. The authors suggest that the observed loss of chromatin structure upon intercalator binding may impact future studies of the mechanism of action of drugs known to act through DNA intercalation.

View bibliography reference

Flagellophora cf. apelti were fixed and permeabilized, and actin was labeled with Alexa Fluor® 488 phalloidin (Cat. no. A12379, green fluorescence). Phospho-H3 was detected using a rabbit primary antibody followed by an Alexa Fluor® 568 dye–labeled goat anti–rabbit IgG antibody (Cat. no. A11011, red fluorescence). Nuclei were stained with blue-fluorescent DAPI (Cat. no. D1306, D3571, D21490). Dr. Smith's laboratory studies stem-cell dynamics in primitive flatworms; Flagellophora belongs to the group Nemertodermatida (arguably the most primitive group of flatworms), whose stem cells (neoblasts) are woefully understudied because the animals are difficult to collect and do not survive long after extraction from sand. The ability to carry out antibody labeling (in this case, anti–phospho-H3 for M-phase cells) is an improvement over traditional methods of S-phase labeling that destroy antigenicity. The image was submitted by Julian Smith, Department of Biology, Winthrop University, USA. 

Find popular products for cell counting and viability, cytotoxicity, and proliferation assays
Reagents for counting cells and measuring cytotoxicity and proliferation are essential research and diagnostic tools. There is great diversity among live cells, their environments, and the physiological and morphological parameters that can be queried during the course of scientific investigation. The assays researchers choose depend on the questions they're asking, the platform they're using, and an evaluation of the advantages and limitations of the various assays that exist. Our new cell health web page highlights some of the most popular and effective products Invitrogen has to offer for assessing cells, and provides an easy way to compare the assay technologies and advantages. Take a look at popular viability, cytotoxicity, and proliferation assays at www.invitrogen.com/cellhealth.

Get your imaging questions answered at ASCB
If you're attending the American Society for Cell Biology (ASCB) meeting in San Francisco, CA, December 13–17, look for Molecular Probes' imaging scientists participating in a "Meet the Scientist" event at the Invitrogen booth (#617). Our scientific support team will be available to discuss your imaging questions throughout the exhibit, and one of our top scientists in signal transduction pathways and kinase biology tools will be available for several hours each day. While you're there, be sure to watch a demonstration of the new Countess™ Automated Cell Counter, and see just how fast and easy cell counting can be. We'll also be highlighting some proven performers, such as the Alexa Fluor® dyes and LIVE/DEAD® assays, and introducing innovative new technologies such as the Click-iT™ EdU cell proliferation assays, and Organelle Lights™ and Cellular Lights™ reagents for easy delivery and multiplexing with fluorescent proteins. Stop by and visit us at booth #617.


Putting the CyQUANT® NF assay to work in drug discovery
Rapid screening of compounds for toxicity—in particular, hepatotoxicity—is a goal for many drug developers. Abraham and coworkers at Abbott Laboratories have developed a means of scoring candidate compounds using a live-cell, multiparametric high-content screening (HCS) assay for hepatotoxicity (Abraham, V.C. et al. (2008) Application of a high-content multiparameter cytotoxicity assay to prioritize compounds based on toxicity potential in humans. J Biomol Screen 13:527–537). The authors used Molecular Probes® stains to assess eight indicators of cell health (see table). By adding measures of mitochondrial total mass and distribution and nuclear aspect ratio and texture, the researchers increased assay sensitivity. The authors were also able to correlate each compound’s mechanism of action with changes in cellular morphology. For example, cell cycle inhibitors, DNA-damaging agents, mitochondrial poisons, and lytic agents can all be readily identified through nuclear swelling, changes in mitochondrial morphology, and plasma membrane permeability, respectively. Based on the HCS data, each compound was assigned a minimum cytotoxic concentration, or IC20 value. The Molecular Probes® CyQUANT® NF assay was used to measure cell proliferation. The researchers were able to prioritize the compounds for further development based on the ratio of minimum cytotoxic concentration to in vitro efficacy IC50/EC50 values. Finding the most sensitive cell health indicator to be cell proliferation, the group compared the higher-throughput CyQUANT® NF assay with a direct HCS measure of cell proliferation. Correlation of the CyQUANT® NF assay with the direct measure of proliferation was very high; thus, the authors recommended using the CyQUANT® NF assay to streamline analysis of cell proliferation. 

Molecular Probes® stains used to assess cell health in the Abbott Laboratories' multiparametric HCS cytotoxicity assay.

  Stain Application Cell health indicator
 YOYO®-1 iodidePlasma membrane integritySignal change measured
 

Tetramethylrhodamine (TMRE)

Mitochondrial functionTotal mass of functional mitochondria
Average brightness of functional mitochondrial mass
Projected area occupied by functioning mitochondrial mass
 

Hoechst 33342

Nuclear morphologyNuclear size
Nuclear aspect ratio
Nuclear texture
 CyQUANT® NF assayCell proliferationCell count

 



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