Alexa Fluor dye–conjugated organelle-specific antibodies: A cellular paint box

By the end of the year, we will offer over 25 different organelle-specific antibodies conjugated to highly fluorescent Invitrogen™ Alexa Fluor™ dyes. These antibodies are monoclonal and polyclonal antibodies that recognize cellular targets typically associated with specific organelles. High-affinity antibodies with specificity for mitochondria, lysosomes, peroxisomes, endosomes, endoplasmic reticulum, cytoskeleton, proteasomes, ribosomes, nucleus, nucleolus, nuclear membrane, cell surface, and cytoplasm have been labeled with the green-fluorescent Alexa Fluor 488 dye, orange-fluorescent Alexa Fluor 555 dye, or far‑red–fluorescent Alexa Fluor 647 dye.

These fluorescent primary antibodies can be directly used in co-localization and other immunofluorescence experiments; no additional secondary detection reagents are required, so multiplex experiments are simplified. In addition to the Alexa Fluor conjugates, the organellespecific antibodies are available unconjugated.

 


Immunofluorescence analysis of ZO-1 in Caco-2 cells. Caco-2 cells were fixed with 4% paraformaldehyde in PBS for 15 min, blocked with 3% BSA in PBS for 30 min, and then stained with the green-fluorescent Invitrogen™ Alexa Fluor™ 488 anti–ZO-1/TJP1 antibody (clone ZO1-1A12) at a dilution of 5 μg/mL in blocking buffer for 1 hr at room temperature, protected from light. Nuclei were counterstained with blue-fluorescent Hoechst™ 33342 dye at a dilution of 1:10,000 in blocking buffer. Images were taken on a Thermo Scientific™ ToxInsight™ instrument at 20x magnification.


T cell and B cell antibody conjugates for flow cytometry

We recently released 20 fluorescent primary antibody conjugates for T cell and B cell targets—including CD3e, CD14, CD35, CD45RA, CD62L, CD56, CD79A, CD80, CD86, CD137, and HLA-DR—available conjugated to the green-fluorescent Alexa Fluor 488 dye or the farred– fluorescent Alexa Fluor 647 dye. Each of these fluorescent antibody conjugates is validated for use in flow cytometry and is protected by the Invitrogen™ antibody performance guarantee (terms and conditions apply).

 


Flow cytometric analysis of CD3e expression in blood cells. Lysed whole blood cells were stained for 20 min with Invitrogen™ Alexa Fluor™ 488 anti–human CD3e antibody (clone UCHT1) and analyzed by flow cytometry, revealing two cell populations based on CD3e detection.


Sensitive, robust SuperSignal West Pico PLUS Chemiluminescent Substrate

Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate is the newest addition to the trusted SuperSignal product line. This enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate enables picogram- to high-femtogram–level protein detection by western blot analysis. Moreover, it is compatible with a variety of membranes and blocking reagents and works exceptionally well over a wide range of antibody dilutions and across many different targets, making it an ideal choice for most western blotting applications.

The innovative, robust formulation of this HRP substrate provides brighter, more intense western blot bands that display strong signal stability for 6 to 24 hours after incubation, allowing more time for multiple exposures to capture publication-quality blot images.

 

Western blot detection using the SuperSignal West Pico PLUS Chemiluminescent Substrate. Detection of the indicated target was performed using 2-fold serial dilutions of HEK 293 (for β-catenin detection) or HeLa (for STAT3 and WNT1 detection) cell lysates, starting at 4 μg/well or 20 μg/well, respectively. Following separation by SDS-PAGE, proteins were transferred to PVDF (for β-catenin) or nitrocellulose (for STAT3 and WNT1) membranes using the Pierce™ Power Blotter and Pierce™ 1-Step Transfer Buffer. The membranes were blocked with 5% nonfat dry milk dissolved in Pierce™ 20X TBS Tween™ 20 Buffer and incubated with antibodies against β-catenin, STAT3, or WNT1, followed by incubation with the HRP conjugate of goat anti–mouse IgG secondary antibody at a concentration of 20 ng/mL. Chemiluminescent detection was performed following a 5 min incubation with Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate. Signal was captured on x-ray film at the indicated time points after addition of substrate.


Mass spectrometry tools for improved quantitation and phosphopeptide enrichment

Thermo Scientific™ TMTsixplex™ and TMT10plex™ Isobaric Label Reagent Sets enable multiplexed protein identification and quantitative analysis by tandem mass spectrometry. Now available in 0.2 mg vials, these amine-reactive isobaric labels are ideal for labeling sample amounts between 10 and 25 μg. The vials are conveniently packaged in an automation-friendly 96-well format, and the single-use format negates the hassle of having to aliquot or discard unused reagent. The caps of the vials have also been color-coded to allow easy identification of the individual sets of isobaric tags.

Thermo Scientific™ High-Select™ Fe-NTA and TiO2 Phosphopeptide Enrichment Kits, which provide fast and efficient enrichment of phosphorylated peptides, have been improved to enable even better selectivity with fewer processing steps. Additional upgrades include better yields for the Fe-NTA phosphopeptide enrichment kit and elimination of the need for clean-up through buffer optimization in the TiO2 phosphopeptide enrichment kit.


New 96-well format and color-coded caps for the TMTsixplex and TMT10plex Isobaric Label Reagent Sets. The Thermo Scientific™ TMTsixplex™ Isobaric Label Reagent Set is shown here.