targeted fluorescent proteins | BacMam delivery | Organelle Lights™ and Cellular Lights™

Organelle Lights™ and Cellular Lights™ targeted fluorescent proteins

what they are
Organelle Lights™ and Cellular Lights™ fluorescent protein–based reagents are targeted to subcellular compartments and organelles by signal sequences. Based on BacMam technology these versatile tools are prepackaged in baculovirus particles for highly efficient, reproducible, and noncytopathic delivery to a range of cell types, including primary and stem cells. Organelle Lights™ and Cellular Lights™ fluorescent proteins are not overtly toxic, which ensures consistent expression. The efficient transduction makes it easy to deliver multiple constructs and to modulate gene expression. Organelle Lights™ and Cellular Lights™ fluorescent proteins are readily adaptable to a number of assay formats and compatibility with automated liquid handling makes them ideal for high-content imaging applications. And now we offer a red fluorescent protein (RFP): TagRFP from Evrogen. TagRFP is a novel monomeric red fluorescent protein based on the wild-type RFP from sea anemone Entacmaea quadricolor (Nat Methods (2007) 4: 555-557). TagRFP is about 3 times brighter than mCherry protein (Nat Biotechnol (2004) 12: 1567-1572), which makes it the brightest monomeric red fluorescent protein currently available.

how they work
Golgi-RFP:
This new Organelle Lights™ product is TagRFP fused to a Golgi targeting tag (a portion of human Golgi-resident enzyme N-acetylgalactosaminyltransferase-2). This is the same tag used in our popular Golgi-GFP.

Nuc-RFP: Our Organelle Lights™ Nuc-RFP comprises TagRFP fused to the SV40 nuclear localization sequence. This allows researchers to visualize the nucleus without utilizing DNA-binding dyes, which may perturb cellular function.

H2B-GFP and -RFP: Another method of localizing the nucleus is via histone labeling. With the Cellular Lights™ H2B-GFP and -RFP products researchers can localize histones in living cells and potentially follow these protein complexes during cell division.

Lysosomes-RFP: A new Organelle Lights™ target, this product is TagRFP fused to the lamp1 protein (lysosomal associated membrane protein 1). With this new product, researchers can now visualize lysosomes, important degradative organelles also involved in autolysis.

Endosomes-GFP: Another new Organelle Lights™ target, we have fused the bright and photostable Emerald GFP to Rab5a, an early endosomal marker. Endosomes-GFP will permit localization of the endosomes within the living cell, and can be fixed as well.MAP4-GFP: One of our most exciting and visually stunning new Cellular Lights™ targets, MAP4 (microtubule associate protein 4) has been fused to Emerald GFP. This product will enable visualization of the microtubules within a living cell.

Tubulin-GFP and -RFP: Complementary to the Cellular Lights™ MAP4-GFP construct is our Cellular Lights™ Tubulin-GFP (Emerald GFP protein fusion) and -RFP (Tag RFP protein fusion) fluorescent proteins. These two products are designed for live-cell imaging of microtubules.

Actin-GFP and -RFP: When added to mammalian cells, our Actin-GFP and Actin-RFP Cellular Lights™ products will produce Emerald GFP or TagRFP fused to the N terminus of beta actin.

Null (control) virus: If the potential effects of baculovirus on your cell type are a concern, our new Cellular Lights™ Null virus lacks any mammalian genetic elements. The Null virus allows you to determine any possible baculovirus-based effects.

 Aortic Smooth Muscle Cells (Cascade Biologics, Invitrogen Cell Culture, C-007-5C) were transduced with Cellular Lights™ MAP4-GFP and Lysosomes-RFP according to the established Molecular Probes® BacMam protocol. The following day Hoechst staining was performed, and cells were imaged using a 63x objective with GFP, RFP, and DAPI filters.

 

 Human Aortic Smooth Muscle Cells (Cascade Biologics, Invitrogen Cell Culture, C-007-5C) were transduced with Cellular Lights™ Tubulin-GFP according to the established Molecular Probes® BacMam protocol. The following day Hoechst staining was performed, and cells were imaged using a 63x objective with GFP and DAPI filters. Nocodazole treatment was performed (10 μM), and images obtained every 2.5 mins for 15 minutes. Nocodazole is an antineoplastic agent that interferes with the polymerization of microtubules.


what they offer

  • intracellular landmarks that are well characterized, accurately targeted, safe and easy to use—no need to make your own constructs and transfection complexes
  • highly efficient transduction of multiple cell types, including primary and stem cells
  • easily "dial in" the right expression level by modulating the dose
  • ideal tools for dynamic live cell studies; compatible with subsequent fixation immunocytochemical processing
  • simple, safe, and fast application of fluorescent proteins with full rights to use

Redox Status | Glutathione Detection | Brighter Thiol Probe

ThiolTracker™ Violet (Glutathione Detection Reagent) for HCS, flow cytometry, and fluorescence microscopy

what it is

ThiolTracker™ Violet is a brighter, more robust, and easier-to-use thiol probe. Ten times brighter than the bimanes conventionally used to detect reduced glutathione, ThiolTracker™ Violet can be used in HCS microplate imaging, flow cytometry, and epifluorescence microscopy. It can be excited with UV or 405 nm laser light and has an emission maximum centered around 525 nm.

how it works

ThiolTracker™ Violet binds intracellular thiols. Because reduced glutathione represents the majority of intracellular free thiols, ThiolTracker™ Violet can be used in estimating the level of reduced glutathione and redox status of a cell. ThiolTracker™ Violet readily crosses the cell membrane, and, upon thiol binding, is transformed into a cell-impermeant reaction product. ThioTracker™ Violet can be fixed by aldehydes, is compatible with Triton® X-100 (0.5%) permeabilization, and is appropriate for multiplex imaging.

what it offers

  • bright signal (10x brighter than bimanes)
  • ease of use
  • consistent, reproducible results
  • can be multiplexed with other assays (for example cytotoxicity studies)
  • useful for glutathione depletion or oxidation—reduction assays

Cell Signaling | Phosphorylation | Primary Antibodies

Phosphorylation site–specific antibodies for protein kinase C isoforms

what they are

Invitrogen offers monospecific reagents for studying the phosphorylation of protein kinase C (PKC), and new additions to this product line cover all major isoforms including Ca++-dependent (α, β, γ), Ca++-independent (δ, ε, η, θ), and atypical (ζ, ι, λ) isoforms

how they work
Phosphorylation is a common mechanism for activation and deactivation of proteins in cell signaling cascades. Phosphorylation site–specific antibodies (PSSA) that are monospecific for the phosphorylation state of a particular tyrosine, serine, or threonine residue on the target protein are critical for elucidating the signaling pathways. Invitrogen offers antibodies against major isoforms of PKC, allowing monitoring of protein expression and regulatory phosphorylation of primary activation sites.

what they offer

  • validated reagents with multiple applications and species specificity
  • wide range of targets and modification sites
  • all phosphorylation site specificities are verified with peptide competition
 Lysates prepared from K562 cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1–4) or treated with lambda (λ) phosphatase (5), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with PKCβI [pT642] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab´)2 anti-rabbit IgG HRP conjugate (Cat. no. ALI4404) and bands were resolved. These data show that only the peptide corresponding to PKCβI [pT642] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phosphospecific.

Mitochondrial Studies | Anti-OxPhos Antibodies | Isolation and Imaging

Monoclonal antibodies for the investigation of mitochondrial structure and OxPhos complexes

what they are

New antibodies, kits, and controls for the study of mitochondrial oxidative phosporylation (OxPhos), including immunocapture and immunocytochemistry kits for the isolation and imaging of OxPhos complexes and specific complex subunits.

how they work
Mitochondria play a crucial role in ATP production through oxidative phosphorylation (OxPhos). OxPhos is carried out by the electron transport machinery, which oxidizes the major products of glycolysis to yield ATP. To accomplish this task, mitochondria have dedicated protein machinery, known as the respiratory protein complexes I, II, III, IV, and V. Invitrogen offers a range of subunit-specific anti–OxPhos Complex mouse monoclonal antibodies that recognize proteins in the oxidative phosphorylation system.

what they offer

  • highly specific monoclonal antibodies with broad validation
  • immunocapture kits and immunocytochemistry kits for imaging
  • the widest selection of OxPhos reagents available

 


See more OxPhos and organelle-specific antibodies and browse antibodies by specificity or application at www.invitrogen.com/antibodies.

Mitochondrial Studies | OxPhos Activity | 96-Well Microplate Assays

MitoProfile® kits for mitochrondrial protein complex analysis

what they are
Invitrogen is now offering assay kits for assaying mitochondrial protein complexes in a 96-well format. The kits can measure mitochondrial protein activity, quantity, or both.

how they work
In the 96-well microplate assay, cytochrome c oxidase is immunocaptured from a sample of cell or tissue extract (no mitochondrial isolation is necessary) by a monoclonal antibody attached to the wells of a 96-well plate. The amount of enzyme captured is determined by the reaction of a second monoclonal antibody against a different epitope in the protein complex with the signal reported by an appropriate secondary antibody, which is conjugated with alkaline phosphatase. In the case of activity microplates, the enzyme is immunocaptured by the plate-bound monoclonal antibody, then its activity measured by following the oxidation of cytochrome c.

what they offer

  • sensitivity—use small sample volume to measure protein levels
  • specificity—measure the correct mitochondria protein of interest
  • multiplex capability—activity and quantity measurements in one well

 

  

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