The new DetectX^® Direct Cyclic AMP (cAMP) Chemiluminescent Immunoassay kit is designed to quantitatively measure cAMP present in lysed cells, tissue plasma, urine, saliva and tissue culture media samples. The kit is unique in that all samples and standards are diluted into an acidic sample Diluent, containing special additives and designed to lyse cells, for measurement. A cAMP standard is provided to generate a standard curve for the assay. A Plate Primer solution is added to all the wells and standards or samples in Diluent are pipetted into the white microtiter plate. cAMP-peroxidase conjugate is added to the standards and samples in the wells. The binding reaction is initiated by the addition of an antibody to cAMP. After 2 hours, the plate is washed and chemiluminescent substrate is added. The substrate reacts with the bound cAMP-peroxidase conjugate. The intensity of the emitted light is detected in a microtiter plate reader capable of measuring luminescence. concentration of cAMP in the sample is calculated, after making suitable correction for the dilutions. An optional acetylation Format allows super low concentrations of cAMP to be measured.

Features

• Measure to less than 1 fmol cAMP per sample - most sensitive kit available
• Measure cAMP in lysed cells, plasma, saliva, urine or tissue
• Colour-coded, simple protocol
• Liquid 4°C stable components
• High signals in 2 hours

 Typical data 

■ Regular format

■ Acetylated format

Sensitivity

Sensitivity was 0.75fmol/well in the Acetylated Format.

Sample preparation

All samples are diluted in the Sample Diluent. Tissue, urine and saliva samples should be run in the Regular Format. Plasma and samples with low expected concentrations of cAMP should be run in the Acetylated Format.
TCM samples should be diluted in TCM and read off a standard curve generated in the same TCM.

Assay protocol

・Pipet 50 µl of Plate Primer into all wells used for the assay.
・50 µl of samples or standards into duplicate wells in the microtiter plate.
・Add 25 µl of diluted DetectX^® cAMP-Peroxidase Conjugate, followed by 25 µ of cAMP Antibody and shake at room temperature for 2 hours.
・Wash 4 times with wash buffer.
・Add 100 µl of chemiluminescent substrate to each well and read each well for 0.1 second.

Linearity

Linearity was determined by taking two Jurkat cell lysate samples, one with a low cAMP level of 0.2 pmol/ml and one with a higher level of 2.5 pmol/ml and mixing them in the ratios given below.

Intra assay precision

Diluted human samples were run in replicates of 20 in both the Regular and Acetylated Formats. The mean and precision of the calculated Cyclic AMP concentrations were:

Inter assay precision

Diluted human samples were run in duplicates in fourteen assays over multiple days by four operators. The mean and precision of the calculated Cyclic AMP concentrations were:

Cross reactivity

Ordering information

Product name	Product No.	Size	Price	Add to cart
High Sensitivity Direct Cyclic AMP Chemiluminescent Immunoassay Kit (without Acetic Anhydride)	K019-C1	1 plate	55,000
	K019-C5	5 plates	221,000

We provide the new products from Tocris Biosciences, Inc., which were added in February 2011.

You can move to the Tocris’ website to know the detail of the products when you click the catalogue number or Product name on the list.

Products	Activity	Tocris' cat#	Size	Price	Add to Cart
UK 356618	Potent and selective MMP-3 inhibitor. Sold for research purposes under agreement from Pfizer Inc. (1)	4187	10 mg	72,500
Torcetrapib	Inhibitor of cholesteryl ester transfer protein (CETP). Sold for research purposes under agreement from Pfizer Inc. (1)	4184	10 mg	64,500
UK 383367	Potent and selective BMP-1 (PCP) inhibitor. Sold for research purposes under agreement from Pfizer Inc. (1)	4188	1 mg	32,500
CP 99994 Dihydrochloride	High affinity NK1 antagonist. Sold for research purposes under agreement from Pfizer Inc. (1)	3417	10 mg	59,500
MCOPPB Trihydrochloride	Potent NOP receptor agonist. Sold for research purposes under agreement from Pfizer Inc. (1)	3763	1 mg	39,500
B-HT 933 Dihydrochloride	Selective α2 agonist.	2758	10 mg	47,500
BINA	Selective positive allosteric modulator of mGlu2.	4048	10 mg	52,500
GSK 9027	Glucocorticoid receptor agonist. Sold for research purposes only under agreement from GlaxoSmithKline. (2)	4116	10 mg	57,500
PD 0325901	Selective inhibitor of MEK1/2. Sold for research purposes under agreement from Pfizer Inc. (1)	4192	10 mg	64,500
SSR 146977 Hydrochloride	Potent and selective NK3 antagonist.	3297	10 mg	59,500
PF 3845	Selective FAAH inhibitor. Sold for research purposes under agreement from Pfizer Inc. (1)	4175	10 mg	64,500
NF 546	Selective P2Y11 agonist.	3892	10 mg	52,500
22-Oxacalcitriol	Non-calcemic vitamin D3 (Cat. No. 4156) analog.	4160	1 mg	47,500
8-Aminoadenine	Adenine receptor agonist.	4050	10 mg	29,500
Polygodial	TRPA1 channel activator; analgesic and antifungal.	3197	10 mg	42,500
Sulprostone	EP3 and EP1 receptor agonist.	3049	1 mg	39,500
Phorbol 12,13-Dibutyrate	Protein kinase C activator.	4153	1 mg	14,500
Tacalcitol	Synthetic vitamin D3 (Cat. No. 4156) analog.	4157	1 mg	57,500
AZD 5438	Potent cyclin-dependent kinase (cdk) 1, 2 and 9 inhibitor. Sold with the permission of AstraZeneca UK Ltd. (3)	3968	10 mg	59,500
Sanguinarine Chloride	Inhibitor of protein phosphase 2C (PP2C).	2302	10 mg	42,500
Salermide	SIRT1 and SIRT2 inhibitor.	4127	10 mg	29,500
Narciclasine	Antiproliferative agent; slows cell cycle progression.	3715	1 mg	39,500
IU1	Usp14 inhibitor.	4088	10 mg	42,500
BMS 753	RARα-selective agonist.	3505	10 mg	47,500
Gisadenafil Besylate	PDE5 inhibitor. Sold for research purposes under agreement from Pfizer Inc. (1)	4165	10 mg	57,500

(1)Sold for research purposes under agreement from Pfizer Inc.
(2)Sold for research purposes only under agreement from GlaxoSmithKline
(3)Sold with the permission of AstraZeneca UK Ltd.

Glycofect is a cationic carbohydrate-containing synthetic polymer based on the　poly-glycoamidoamine (PGAA) polymer library developed by Dr. Theresa M. Reineke et al. These polymers contain linkages that hydrolyse upon endocytosis, thereby increasing plasmid delivery and reducing toxicity. Glycofect is a superior reagent for primary cell transfection as compared to leading lipid and poly-cationic based products.

Documented Results-1

The following micrographs compare Glycofect to leading lipid-based reagents when used to transfect primary dermal fibroblasts and primary aortic smooth muscle cells. In each series, the DIC images (top row) show the density of the individual cells while the fluorescence iimages (bottom row) show those cells that have been transfected with a plasmid expressing green fluorescent protein. All samples were prepared per manufacturers' instructions.
■ Primary Dermal Fibroblasts

■ Primary Human Aortic Smooth Muscle

Documented Results-2

■ Primary Dermal Fibroblasts

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This is a stain solution kit that blackens polysaccharide components of fungal cell wall by using chromic acid and alkaline hexamine-silver complex. Chromic acid oxidizes polysaccharides and forms aldehydes. Then aldehydes reduce alkaline hexamine-silver complex, and the color of cell walls of fungi turns to black.
This solution kit is useful for both room temperature and microwave method.

Results

Ordering information

Product name	Product No.	Size	Price	Add to Cart
Grocott Methenamine Silver Stain (GMS) for Fungus and PCP - Room Temperature & Microwave Kit	25087-1	1 kit	70,000

We provide the new products from Tocris Biosciences, Inc., which were added in November 2010.

You can move to the Tocris’ website to know the detail of the products when you click the catalogue number or Product name on the list.

We provide the new products from Tocris Biosciences, Inc., which were added in October 2010.

You can move to the Tocris’ website to know the detail of the products when you click the catalogue number or Product name on the list.

Tocris' cat#	Products	Activity	Size	Price	Add to Cart
3854	1-Oleoyl lysophosphatidic acid sodium salt	Endogenous agonist of the lysophospholipid receptors LPA1 and LPA2. Inhibits differentiation of neural stem cells	1mg	9,500
2430	GGTI 298	Geranylgeranyltransferase I (GGTase I) inhibitor	1mg	44,500
3516	Tunicamycin	Antibiotic; GlcNAc phosphotransferase inhibitor	10mg	42,500
3897	API-1	Selective Akt/PKB inhibitor. Antitumor	10mg	77,500
3989	AMTB hydrochloride	TRPM8 blocker	10mg	49,500
			50mg	219,500
4059	[Glu27]-PKC (19-36)	Inactive control peptide for PKC (19-36), a pseudosubstrate peptide inhibitor of protein kinase C.	1mg	32,500
3795	TUG 424	Agonist of the free fatty acid receptor FFA1 (GPR40). Sold under license from the University of Southern Denmark(1).	10mg	44,500
			50mg	192,500
3998	LDN 57444	Ubiquitin C-terminal hydrolase-L1 (UCH-L1) inhibitor	10mg	32,500
			50mg	139,500
3360	CGP 52411	Selective inhibitor of the epidermal growth factor receptor (EGFR).  Also inhibits the formation of Aβ42 fibers associated with Alzheimer's disease.	10mg	37,500
			50mg	164,500
2766	Naspm trihydrochloride	Ca2+-permeable AMPA receptor antagonist	10mg	240,000
3743	BF 2649	H3 receptor inverse agonist/antagonist	10mg	37,500
			50mg	164,500
3893	S 107 hydrochloride	Type 1 ryanodine receptor (RyR1) stabilizer; inhibits Ca2+ leak from the sarcoplasmic reticulum (SR)	10mg	42,500
			50mg	174,500
3947	exo-IWR 1	Negative control for endo-IWR 1 (Cat.No. 3532)	10mg	34,500
			50mg	147,500
4025	α-Amanitin	Inhibitor of RNA polymerase II	1mg	62,500
4042	PSC 833	P-glycoprotein (P-gp) modulator, Inhibitor of P-gp -mediated multidrug-resistance (MDR)	1mg	54,500
3829	UK 78282 hydrochloride	Blocker of KV1.3 and KV1.4 channels  in T lymphocytes	10mg	52,500
			50mg	227,500
3759	Exemestane	Irreversible steroidal aromatase (CYP19) inhibitor. Sold for research purposes under agreement from Pfizer Inc(2).	10mg	42,500
			50mg	174,500
3761	Tolterodine L-tartrate	Potent, non-selective muscarinic receptor antagonist. Sold for research purposes under agreement from Pfizer Inc(3).	10mg	32,500
			50mg	139,500
2918	Veratridine	Voltage-gated Na+ channel opener	10mg	24,500
			50mg	104,500
3921	NIDA 41020	High affinity CB1 antagonist	10mg	47,500
			50mg	202,500
3831	LG 100754	Novel RXR:PPARγ agonist	10mg	57,500
			50mg	244,500

The LEXSY in vitro translation system is a NEW rapid, convenient, flexible and cost efficient tool for cell-free production of recombinant proteins for biochemical, biophysical and structural analysis.
The LEXSY cell extract for in vitro translation contains functional ribosomes and all essential components of the eukaryotic translation and folding machinery. For target mRNA generation the heterologous T7 RNA polymerase was added to the extracts. To ensure a low background the translation of endogenous host mRNAs is efficiently blocked by an antisense oligonucleotide making use of the unique gene organization of Leishmania.

Vector map

Map of pLEXSY_invitro-1 (Cat.-No. EGE-261) expression vector with cloning sites for target gene in frame fusions. 3’ utr is derived from 1.4k-IR camCB of L. tarentolae. MCS1 and MCS2 are multicloning sites for replacement of vector EGFP control gene or N’ or C’ fusions resp. SITS is species independent translation sequence (Mureev et al., 2009).

Kit components

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ProAssay Kits use FRET principle and are for not only measuring the activity of proteases but also high-throughput screening of protease inhibitors.

HCV Protease Assay Kit

The HCV NS3/4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (1-4). The order and kinetics of cleavage as well as the extent of precursor processing appear to be critical steps in the generation of fully infectious, appropriately assembled viral particles. Therefore, inhibition of HCV NS3/4A protease represents an important avenue for antiviral therapy (2).

ProAssay HCV Protease Assay Kit provides a convenient method for high throughput screening of HCV NS3/4A protease inhibitors and continuous quantification of HCV protease activity using FRET peptide substrate. The sequence of this FRET peptide is derived from NS3-dependent cleavage site:Asp/GluXaa4Cys/Thr-Ser/Ala (5). In the FRET peptide, the green fluorescence is quenched by appropriate fluorescence quencher until this peptide is cleaved into two separate fragments by HCV NS3/4A protease at the cleavage site. Upon cleavage, the green fluorescence is recovered and can be monitored at excitation/emission = 490 nm/530 nm.

HIV-1 protease Assay Kit

HIV-1 protease (HIV-1 PR) is responsible for cleaving up to 12 sites in the Gag and Gag-Pol precursor polypeptides (6, 7). The order and kinetics of cleavage as well as the extent of precursor processing appear to be critical steps in the generation of fully infectious, appropriately assembled viral particles (8-11). Therefore, inhibition of HIV-1 protease represents an important avenue for antiviral therapy (11).

ProAssay HIV-1 Protease FRET Assay Kit provides a convenient method for high throughput screening of HIV-1 protease inhibitors and continuous quantification of HIV-1 protease activity using a fluorescence resonance energy transfer (FRET) peptide. The sequence of this FRET peptide is derived from the native p17/p24 cleavage site on Prgag for HIV-1 protease. In the FRET peptide, the green fluorescence is quenched by appropriate fluorescence quencher until this peptide is cleaved into two separate fragments by HIV-1 protease at the cleavage site. Upon cleavage, the green fluorescence is recovered and can be monitored at excitation/emission = 490 nm/530 nm.

Principle of the detection

The cleavage site locates between the quencher and fluorophore on the substrate peptide, and the fluorescence of fluorophore is quenched by the quencher. When proteases cut the cleavage site, the peptide cleaved into 2 flagment, so that the fluorescence is recovered and can be monitored (ex / em = 490 nm / 530 nm).

Kit Components

References

1. Grakoui, A. et al. J Virol. 67, 2832-2843. (1993)
2. Hijikata, M. et al. Proc Natl Acad Sci U S A. 90, 10773-10777. (1993)
3. Tomei, L. et al. J Virol. 67, 4017-4026. (1993)
4. Lamarre, D. et al. Nature 426, 186-189. (2003)
5. Talianie, M. et al. Anal Biochem. 240, 60-67. (1996)
6. Chou, KC. et al. Proteins 24, 51-72. (1996)
7. Henderson, LE. et al. In: Bolognesi, D (ed.), Human Retroviruses, Cancer and AIDS: Approaches to Prevention and Therapy. Alan R. Liss, New York, 135-147. (1988)
8. Krausslich, HG. et al. J Virol. 69, 3407-3419. (1995)
9. Mervis, RJ. et al. J Virol. 62, 3993-4002. (1988)
10. Pettit, SC. et al. J Virol. 68, 8017-8027. (1994)
11. Temesgen, Z. Expert Opin Pharmacother. 2, 1239-1246. (2001)

Ordering Information

DiUbiquitin IQF Substrate

Deubiquitylases (DUBs) are proteases that are responsible for the release of ubiquitin or ubiquitin chains from target proteins in vivo.  This activity requires the cleavage of an isopeptide bond between the terminal glycine of ubiquitin and the e-amino group of a lysine residue in the target.  Until now, all high throughput compatible deubiquitylase assays have measured the cleavage of a linear amide or peptide bond between ubiquitin and either a small fluorophore (e.g. AMC, Rhodamine110) or a reporter protein (CHOP assays).  LifeSensors’ new IQF-DiUb™ substrates are the first commercially available deubiquitylase substrates that permit continuous monitoring of isopeptide bond cleavage in a homogeneous format.  This is achieved by incorporating a fluorescent dye on one ubiquitin and an appropriate fluorescence quenching dye on the second, resulting in an internally quenched fluorescence (IQF) molecule.  Cleavage of the isopeptide bond linking the ubiquitin subunits results in an increase in fluorescent signal when the quencher and fluorophore are separated.  In order to minimize perturbation of ubiquitin’s structure, the dyes are attached to specific sites within the ubiquitin molecule and not at the N- or C- termini.  Since some labeling sites may affect recognition by any given DUB, we have created a panel of different diubiquitin molecules within each linkage class using several different labeling sites, e.g. we offer six different versions of our K48 linked diubiquitin.  We recommend that you start with the survey panel of diubiquitins in order to determine which version of a particular linkage class gives the optimal results under your assay conditions in terms of sensitivity, signal to background, and turn over by your DUB.

The generation of ubiquitin isopeptidase assays using physiologically relevant substrates represents a major advancement in the study of this important group of cellular enzymes.

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The conjugation of ubiquitin to substrates is a multi-step, multi-enzyme process. LifeSensors is committed to developing high throughput, facile, and robust assays for measuring the activity of ubiquitin conjugating enzymes. LifeSensors has recently developed a microtiter plate-based assay for measuring protein ubiquitylation. This assay is amenable to high throughput screening and works with a wide range of E3 ligases including members of the three major classes of E3 ligases (simple RING class, SCF RING class, and HECT class)

E3LITE Customizable Ubiquitin Ligase Kit / E2 Profiling Kit

They are essentially providing a singular platform for the focused investigation of any E2/E3 enzyme pair, as well as any particular polyubiquitylation linkage type. In support of this flexible platform, LifeSensors offers the widest array of reagents on the market to meet your research needs. The kit itself can be assembled with any one of more than E2 conjugation enzymes, with a selection representing members from each enzyme class. LifeSensors also offers E2 Selection Kits that provides a panel of (4) E2s. The recombinant ubiquitin component of this kit can be wild-type, variants that specifically form K48 linkage types, or variants that form linkages only through one of the seven possible surface exposed lysines.

More Information

• E2 Profiling Kit
• E3LITE Customizable Ubiquitin Ligase Kit

E3 Substrate Identification Array Support Kit

This kit provides reagents and protocols for array-based discovery of E3 substrates, including all components necessary for substrate ubiquitylation as well as LifeSensors' proprietary detection reagent for polyubiquitin chains.  The user must provide a protein microarray and FITC conjugated avidin/streptavidin. LifeSensors has developed an extremely sensitive poly-ubiquitin detection reagent with nanomolar affinity for poly-ubiquitin chains. Utilizing this detection reagent in combination with protein microarrays allows the user to identify potential E3 ligase substrates.

More Information

• E3 Substrate Identification Array Support Kit

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TUBE (Tandem Ubiquitin Binding Entity)(LifeSensors)

Example

■ Pulldown of Total Polyubiquitinated Protein with Agarode-TUBE1

Approximately 5mg of total protein was isolated from HEK293T cells and subjected to pulldown with 10 µl of Agarose-TUBE 1. Input and Flow-through (1/10th of total) are shown for comparison. Protein was probed with anti-ubiquitin lgG.

We provide the new products from Tocris Biosciences, Inc. which were added in July 2010.

You can move to the Tocris’ website to know the detail of the products when you click the Product Name on the list.

NOTE: Tocris Biosciences, Inc. sells AR-A 014418 (# 3966) with the permission of AstraZeneca UK Ltd.

(1) Colony screening "by eye" using a fluorescent reporter

The inducible protein expression platform LEXSY is based on integration of an expression cassette (containing a protein of interest) into a chromosomal locus of the host under control of a T4 RNA polymerase / TET repressor architecture.

Our new vector generation now allows convenient co-expression of the protein of interest with BleCherry conferring both, antibiotic resistance for selection as well as visible fluorescence under daylight as a direct read out for protein production.

Upon plating on solid media, expressing clones are identified "by eye" by their cherry color at daylight (Fig. 1), whereas flawed clones (gray color) can be excluded from further analysis.

Figure 1: Picture of induced LEXSY clones co-expressing the BleCherry reporter.

(2) Direct read out for protein production

The BleCherry fluorescence is an indicator for integrity of the system and its expression capabilities. Small samples (0.1 - 1 ml) are taken from the culture for measuring their cherry fluorescence. This allows post-inductional monitoring of expression and thereby optimization of the point of harvest (Fig. 2).

Figure 2: Cherry fluorescence of LEXSY cultures (#1-5) 48 and 72 h post induction. Samples of 1 ml were taken and fluorescence was determined (ex 590 nm / em 620 nm) allowing i) monitoring the time course of expression for individual cultures and ii) comparison of expression levels for various cultures [- (No Tetracycline), + (Tetracycline 10 mg/l)], H host control.

View the illustration of the 3rd generation of inducible LEXSY

( http://www.jenabioscience.com/images/b3e879b381/Illustration_ind_LEXSY_blecherry_EGE-1410__100625.pdf?utm_source=newsletter_jul10&utm_medium=E-Mail&utm_campaign=LEXSY%2BJul%2B2010 )

(3) Minimized vectors for easier cloning

In addition to the above, the new pLEXSY_I-3 vector generation has been reduced in size by about 1 kb compared to the previous vectors which allows easier cloning of large target genes (> 5 kbp) for expression.

(4) LEXSY products – all you need for cloning, expression, strain selection, and cultivation

LEXSY Expression Kits

Convenient all-in-one expression kits allowing constitutive or inducible, intracellular or secretory expression of target proteins

LEXSY Strains & Cultivation Kits

The LEXSY expression hosts, as strains or complete ready-to-grow cultivation kits.

LEXSY Media, Antibiotics, Additives

Everything for growth and maintenance of strains.

LEXSY Molecular Tools

Expression vectors and useful primers.

PCR and Cloning enzymes

All restriction enzymes for the pLEXSY-2 cloning sites, ligases, polymerases etc...

Need further information? Please check out the LEXSY section at the Jena Bioscience website!

( http://www.jenabioscience.com/cms/en/1/browse/1007/?utm_source=newsletter_jul10&utm_medium=E-Mail&utm_campaign=LEXSY%2BJul%2B2010 )