ProbesOnline

• Take advantage of the best specificity for mitochondrial proteins
• Take a closer look at phosphorylation
• Fast, specific protein quantification
• Simultaneously measure 10 markers in one well
• A third color for your flow cytometer
• Shedding new light on lipid homeostasis
• buzzworthy
• the view
• on the web
• in the field

Primary antibodies against mitochondrial Complex III and Complex IV subunits

what they are
Invitrogen offers antibodies against subunits of cytochrome c, Complex III (cytochrome c reductase), and Complex IV (cytochrome c oxidase), validated against multiple species in multiple applications. Invitrogen now provides the most complete portfolio of antibodies for Complex III, Complex IV, and cytochrome c.

how they work
Genetic alterations in Complex III and Complex IV are a common cause of many OxPhos genetic diseases. These complexes are members of the electron transport chain in mitochondria, and deficiencies in these complexes result in many genetic neurological diseases. In addition, deficiencies in OxPhos and electron transport can result in conditions such as Alzheimer’s disease, Parkinson’s disease, diabetes, and cancer.

what they offer

• best specificity for mitochondrial proteins
• validated reagents with multiple applications and species specificities
• part of a wide-ranging portfolio of antibodies for mitochondrial research

Localization of cytochrome c in HDFn cells. Cytochrome c is shown in red after treatment with Anti-Cytochrome c Monoclonal Antibody (Cat. no. 456100) and Alexa Fluor® 594 goat anti–mouse IgG secondary antibody. Nuclei are shown in blue.

Products	Reactivity	Application	Size	Cat. no.
Complex III subunit core 1, OxPhos, Monoclonal Antibody	Hu, B, Ms , Rt	WB	100 µg	459140
Complex III Immunocapture Monoclonal Antibody	Hu, B, Rt, Ms, Z, Dm, Ce	IP	100 µg	439400
Complex IV subunit Vb, OxPhos, Monoclonal Antibody	Hu, B, Ms , Rt	WB	100 µg	459110
Complex IV subunit Va, OxPhos, Monoclonal Antibody	Hu, B, Ms, Rt, Z	WB	100 µg	459120
Complex IV subunit II, OxPhos, Monoclonal Antibody	Sc	WB	100 µg	459150
Complex IV III-subunit Monoclonal Antibody	Hu, Ms, B, Rt, Ce	WB	100 µg	459300
Complex IV subunit I, OxPhos, Monoclonal Antibody	Hu, Ms, B, Rt, Ce, Z, Oyster	WB	100 µg	459600
Complex IV Immunocapture Monoclonal antibody	Hu, B, Ms	IP	100 µg	439500
Complex IV subunit SURF1 Monoclonal antibody	Hu	WB	100 µg	439600
Cytochrome c Monoclonal Antibody	Hu	WB, ICC	100 µg	456100
Cytochrome c WB Cocktail ApoTrack™	Hu	WB	100 µg	459160
Complex IV Mitoprofile® Immunocapture Kit	Hu, B, Ms, Rt	IP	100 µg	457225
Complex IV Mitoprofile® Immunocapture Kit for Rodent	Hu, B	IP	250 µg	457325
Complex III Mitoprofile® Immunocapture Kit	Hu, B, Ms, Rt	IP	250 µg	457125
Reactivity: Hu = human; B = bovine; Ms = mouse; Rt = rat; Z = zebrafish; Dm = D. melanogaster; Ce = C. elegans; Sc = S. cerevisiae. Application: WB = western blot; IP = immunoprecipitation; ICC = immunocytochemistry.

For a complete list of mitochondrial research antibodies, visit www.invitrogen.com/antibodies.

Phosphorylation site–specific antibodies for retinoblastoma protein (Rb)

what they are
Invitrogen offers the highest-quality phosphorylation site–specific antibodies for studying phosphorylation of retinoblastoma (Rb) at various sites, as well as pan antibodies for detection of total Rb.

how they work
Differential phosphorylation of Rb plays a pivotal role in cell cycle regulation. Phosphorylation of Rb affects many cell cycle pathways, which in turn play a part in the development and progression of many tumors. Invitrogen's highly specific antibodies against Rb are the perfect tools for cell cycle, cancer, and general cellular research. Invitrogen offers the best portfolio of highest-quality antibodies for cell signaling research. Each phosphorylation-specific antibody is affinity purified and negatively adsorbed, with specificity verified using peptide competition for each production lot.

what they offer

• validated reagents with multiple applications and species specificities
• wide range of targets and modification sites
• all phosphorylation-site specificities verified with peptide competition

Western blot showing peptide blocking of retinoblastoma (Rb) [pSpS807/811]. MCF-7 cells were synchronized in EMEM (+glutamine and fungal P/S) with sodium selenite, transferrin, and 10% FBS treated with dextran and charcoal for 3 days followed by the above medium without serum for 24 hr. The cells were released with 2 ng/ml IGF-1 and 10% FBS added to EMEM. Cell extracts were made at 0 hr, 8 hr, 16 hr, 20 hr, 24 hr, and 28 hr time points. The time points shown here most clearly represent the upregulation of phosphorylation. To verify that the upregulation of the signal is due to recognition of the phosphorylated form of Rb, peptide-blocking studies were undertaken. Membranes were incubated with pan Rb antibody, clone 1F8 (Cat. no. AHO0212) at a 1:1,000 dilution (1–4). A transition from the hypo- to hyperphosphorylated forms is evident by the two sets of multiple bands between the two forms. The membranes were then incubated with 0.5 µg/ml Rb [pSpS807/811] antibody, following prior incubation with no peptide (lanes 5–8), the nonphosphopeptide corresponding to the immunogen (lanes 9–12), or the phosphopeptide immunogen (lanes 13–16). After washing, membranes were incubated with goat F(ab’)₂ anti–rabbit IgG alkaline phosphatase (Cat. no. ALI4405), and bands were detected using the Tropix WesternStar™ detection method. The antibody only recognizes the phosphorylated form of Rb as the cells progress through the cell cycle. The data also show that only the peptide corresponding to Rb [pSpS807/811] blocks the antibody signal, demonstrating the specificity of the antibody.

Description	Reactivity	Applications	Size	Cat. no.
RB [pSpS807/811] pAb glyc	Hu, Ms	WB	10 blot	44-580G
RB [pT356] PAb	Hu	WB	10 blot	44-578
RB [pS608] PAb	Hu	WB, IP, E	50 μg	34-1800
RB [pS612] PAb	Hu	WB	10 blot	44-572
RB [pS780] PAb	Hu	WB	10 blot	44-574G
RB [pS807] PAb	Hu	WB	10 blot	44-579
RB [pT821] PAb	Hu	WB	10 blot	44-582G
RB [pT826] PAb	Hu	WB	10 blot	44-576G
RB [pSpT249/252] PAb	Hu	WB	10 blot	44-584G
RB [pSpS807/811] PAb	Hu, Ms	WB	10 blot	44-580
RB (XZ55), Mouse Monoclonal	Hu, Ms, Ch, Xe, Quail	IP, WB	100 μg	AHO0182
RB Mouse Monoclonal	Hu, Ms, Ch	IP, IHC	100 μg	AHO0212
RB (1F8 OR RB1), Mouse Monoclonal	Hu	WB, IHC, IP, FC	100 μg	AHO0172
RB (XZ104) Mouse Monoclonal	Hu, Ms, Ch	IHC, IF, IP	100 μg	AHO0202
Reactivity: Hu = human; Ms = mouse; Ch = chicken; Xe = Xenopus. Application: WB = western blot; IP = immunoprecipitation; ICC = immunocytochemistry; FC = flow cytometry; IF = immunofluorescence; IHC = immunohistochemistry; E = electrophoresis.

For a list of additional phosphorylation site–specific antibodies, visit www.invitrogen.com/pssa. To browse antibodies by specificity or application, visit www.invitrogen.com/antibodies.

ELISA kits for ACC1 [pS79] and total ACC1 protein

what they are
Invitrogen’s new ELISA (enzyme-linked immunosorbent assay) kits measure ACC1 phosphorylated at serine 79 as well as total ACC1 protein levels in human cell lysates. Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that catalyzes the ATP-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, a pivotal step in the fatty acid synthesis pathway. ACC activity is tightly regulated through a variety of dietary, hormonal, and other physiological responses. The activity of ACC is inhibited when it is phosphorylated. Recent findings have suggested the concept of direct inhibition of ACC activity as an important therapeutic target against a variety of human diseases, including diabetes, obesity, cancer, and microbial infections.
 
how they work
The new Invitrogen™ ELISA kits are solid-phase sandwich ELISAs that provide a simple and unbiased way to quantify specific proteins. These kits are provided ready to use with all the necessary reagents, including calibrated standards. The 96-well plate included in the kit is precoated with antibodies; when a sample is added to a well, the immobilized capture antibody binds to the protein antigen. Nonspecific binding is washed away. A detector antibody and a secondary antibody conjugated to horseradish peroxidase (HRP) are then added, as well as a chromogen substrate. The chromogen substrate reacts with any bound enzyme to produce a colored product, which is directly proportional to the concentration of the protein present. Finally, stop solution is added to terminate the color reaction, and intensity is measured using a plate reader.

what they offer

• sensitivity—two times more sensitive than western blotting
• specificity—measure the correct protein of interest
• speed—four hours to complete

Specificity testing. HepG2 cells were treated with AICAR (2 µM). Untreated HepG2 cells were used as a control. Cell extracts were prepared in Cell Extraction Buffer (Cat. no. FNN0011). 50 ml of each cell lysate (200 µg/ml) was analyzed with human ACC1 [pS79] ELISA (Cat. no. KHO1061) and human ACC1 (Total) ELISA (Cat. no. KHO1071). The results show that the phosphorylation of ACC1 is upregulated in AICAR-treated HepG2 cells, whereas the level of ACC1 (Total) remains approximately the same in AICAR-treated and untreated HepG2 cells.

Product	Quantity	Cat. no.
ACC1 [pS79] phosphoELISA™ Kit	96 tests	KHO1061
ACC1 Total ELISA Kit	96 tests	KHO1071

For a complete list of ELISA assays, visit www.invitrogen.com/elisa.

Cytokine Rat 10-Plex Panel

what it is
The Invitrogen™ Cytokine Rat 10-Plex Panel is designed for the in vitro quantitative determination of ten analytes. This kit provides a series of combined reagents for the simultaneous measurement of ten rat cytokines, including GM-CSF, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α. The 10-plex panel can also be combined with other Invitrogen™ rat extracellular bead reagents, allowing greater multiplexing of the assay. Samples may include serum, plasma, or tissue culture supernatant.

how it works
The Cytokine Rat 10-Plex Panel is a solid-phase sandwich immunoassay designed for simultaneously quantifying the levels of multiple biomarkers in rat samples. Beads of defined spectral properties conjugated to analyte-specific capture antibodies and samples (including standards of known analyte concentration, control specimens, and unknowns) are pipetted into the wells of a filter-bottom microplate and incubated for 2 hr. During this first incubation, analytes bind to the capture antibodies on the beads. After washing the beads, analyte-specific biotinylated detector antibodies are added and incubated with the beads for 1 hr. During this second incubation, the analyte-specific biotinylated detector antibodies recognize their epitopes and bind to the appropriate immobilized analytes. After removal of excess biotinylated detector antibodies, streptavidin conjugated to the fluorescent protein R-phycoerythrin (streptavidin-RPE) is added and incubated for 30 min. During this final incubation, the streptavidin-RPE binds to the biotinylated detector antibodies associated with the immune complexes on the beads, forming a four-member solid-phase sandwich. After washing to remove unbound streptavidin-RPE, the beads are analyzed using the Luminex® 100™ or 200™ instrument.

what it offers

• superior performance—accurate, reproducible, and sensitive quantitation of multiple proteins
• high quality—in-house manufactured antibodies ensure excellent specificity and sensitivity
• fast and easy protocols—perform the assay and analyze your data in less than a day

Cytokine production profile of a rat splenocyte detected by the Cytokine Rat 10-Plex Panel.  A rat splenocyte was stimulated with PMA at 50 ng/ml and calcium ionophore (Cat. no. A23187) at 250 ng/ml at 24 hr, 48 hr, and 72 hr. The supernatant was collected and used in a multiplex bead assay.

Product	Quantity	Cat. no.
Cytokine Rat 10-Plex Panel	100 tests	LRC0002

For a complete product listing of multiplex Luminex® assays, visit www.invitrogen.com/luminex.

PerCP conjugates for flow cytometry

what they are
Invitrogen now offers primary antibodies conjugated to peridinin chlorophyll protein (PerCP) that are validated for flow cytometry.

how they work
PerCP is a fluorochrome commonly used as a third color on 488 nm laser–equipped flow cytometers. PerCP is excited by the 488 nm laser, with peak emission at 678 nm, corresponding to FL3 on the FACScan™, FACSCalibur™, and FACSCanto™ cytometers (BD Biosciences), and FL4 on Beckman Coulter instruments. Invitrogen now offers direct conjugates of PerCP primary antibodies for flow cytometry applications.

what it offers

• a third color option for single-laser cytometers
• excitation with the 488 nm laser, and detection in the red channel (FL3 on most instruments)
• matching isotype controls

Human peripheral blood lymphocytes were stained with PerCP-conjugated anti–human CD8 monoclonal antibody (Cat. no. MHCD0831, clone: 3B5). The negative control profiles represent unstained cells.

Human peripheral blood monocytes were stained with PerCP-conjugated anti–human CD14 monoclonal antibody (Cat. no. MHCD1431, clone: TüK4). The negative control profiles represent unstained cells.

Human peripheral blood lymphocytes were stained with PerCP-conjugated anti–human CD16 antibody (Cat. no. MHCD1631; clone: 368) to delineate natural killer cells. The negative profile represents unstained cells.

Product	Regulatory status	Quantity	Cat. no.
Mouse anti-human CD4 PerCP	ASR	0.5 ml	MHCD0431
Mouse anti-human CD8 PerCP	ASR	0.5 ml	MHCD0831
Mouse anti-human CD14 PerCP	ASR	0.5 ml	MHCD1431
Mouse anti-human CD16 PerCP	ASR	0.5 ml	MHCD1631
Mouse anti-human CD45RO PerCP	ASR	0.5 ml	MHCD45RO31

To browse antibodies by specificity or application, visit www.invitrogen.com/antibodies.
To find antibodies and other reagents for flow cytometry, visit www.invitrogen.com/flowcytometry.

PLA1 and PLA2 ready-to-use assays

what they are
As researchers examine the role of phospholipases in atherosclerosis, Alzheimer’s disease (AD), and other conditions, Invitrogen’s fluorescence-based kits for PLA1 and PLA2 detection are making these studies easier and more economical.

how they work
Cholesterol disregulation is a risk factor in AD, diabetes, multiple sclerosis (MS), Huntington’s disease, and other conditions. With an aging population aware that 20% of those over age 80 will be diagnosed with AD, and with 50 million Americans suffering from metabolic disorder (characterized by excessive belly fat, hypertension, hypoglycemia, and high LDL levels), cholesterol remains a research focus.

In 1973, Brown and Goldstein found that a genetic defect that elevates the action of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) also causes familial hypercholesterolemia. In 2008, inhibitors of this enzyme, collectively called statins, are prescribed to more than 35 million Americans. For patients at risk for cardiovascular disease (CVD), which is estimated to kill 22 million worldwide, statin drugs have lowered LDL concentrations and elevated HDL levels.

But there is growing controversy that merely lowering cholesterol is protective for many, but not all, risk groups. Because statin therapy carries risks, other solutions are being considered. Phospholipases 1 and 2 (PLA1 and PLA2) are high on the list of potential drug targets. Expression of PLA1 and PLA2 can not only alter LDL and HDL levels, but also increase their atherogenicity by raising their free cholesterol and fatty acid content. These lipids normally exist together as esters, but the action of lipases can expose toxic forms, and PLA1 may, in some cases, completely metabolize HDL particles. Many types of PLA2 exist, defined as those enzymes which cleave at the SN2 site in the phospholipid head groups. However, it is the secreted forms—in particular, those found on LDL itself—whose overexpression is seen as predictive of CVD, with many researchers suggesting that PLA2 will soon replace C-reactive protein as the go-to patient diagnostic test. PLA1, a newcomer that cleaves at the SN1 site, is expressed on the surface of endothelial cells, where it is tethered by a heparin sulfate proteoglycan linkage. Mice lacking the PLA1 gene have elevated levels of HDL. Interestingly, the liver can express a protein in LDL, paraoxonase, that can protect against lipase actions. A clear understanding not just of serum cholesterol levels, but of their status, offers a more complete approach to treating lipid disregulation disease.

It has been shown that inflammation increases lipase expression, and NSAIDs have been shown to decrease their activity. NSAIDs have already been shown to reduce AD risk by 4-fold, and a new generation of NSAIDs that offer greater specificity for these enzymes are being sought. Lipases now hold promise for treatment of CVD and AD, with the possibility that one drug could lower the risk for both disease states.

Invitrogen offers many assays, antibodies, and other tools for studying lipid regulation, including high-throughput screening (HTS)–ready assays for paraoxonase, myeloperoxidase, labeled LDL particles, cholesterol, and now PLA1, PLA2, and phosphatidylcholine-specific phospholipase C assays in kit form. All three lipase kits are simple mix-and-read formulations that allow continuous monitoring in kinetic or endpoint assays. Cleavage of the new PLA1 and PLA2 substrates results in a 20-fold signal increase in 96-well format. The robustness and scalability of these reagents were well established in a recent study (J Lipid Res (2007) 48:472) using a nonspecific, less-fluorogenic lipase substrate, bis-BODIPY (B7701). In that study, the researchers were able to scale the assay to ultra-throughput, 1,536-well plates. The new PLA1 and PLA2 substrates have improved the signal gain at least 5-fold over that of bis-BODIPY. In addition, by altering the SN1 and SN2 linkages, the substrates are selective for PLA1 and PLA2. The PL-C assay is a direct, non-enzyme-coupled assay that is now offered in kit form. The kits contain all lipids and buffers needed to run the assays.

References
 Proc Natl Acad Sci USA (1973) 70:2804; Nature (2008) 451:904; Arch Neurol (2007) 64:93; J Internal Medicine (2006) 260:211; J Lipid Res (2007) 48:472; Curr Drug Targets (2007) 8:1307; Circulation (2008) 117:678.

what they offer

• ready-to-use assays for PLA1 and PLA2
• direct, noncoupled assay for phosphatidylcholine-specific PL-C
• enyzme controls and buffers supplied
• fluorescence-based readouts for higher sensitivity
• freedom from mass spectrometry and assays that require radioactivity and antibodies
• screens at pennies per well

phospholipase C, A1, and A2 (PLA1 and PLA2)

Product	Quantity	Cat. no.
EnzChek® Phospholipase A1 Assay Kit	2 to 10 plates	E10219
EnzChek® Phospholipase A1 Assay Kit	10 to 100 plates	E10221
EnzChek® Direct Phospholipase C Assay Kit	2 plates	E10215
EnzChek® Direct Phospholipase C Assay Kit	10 plates	E10216
EnzChek® Phospholipase A2 Assay Kit	2 plates	E10217
EnzChek® Phospholipase A2 Assay Kit	10 plates	E10218