emd4biosciences newsletter issue 1 2010 - a calbiochem & novagen product brand newsletter

8/8/2019 EMD4Biosciences Newsletter Issue 1 2010 - A Calbiochem & Novagen Product Brand Newsletter

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No. 1 | July 2010

Karl Willert, Ph.D., Director StemCell Core Facility, University of California, San Diego, USA

INTRODUCTIONThe derivation of the first human embry-onic stem cells (hESCs) by Dr. JamesThomson in 1998 1 ushered in a new era inbiomedical research and opened the door to novel therapeutic treatments for previ-ously incurable diseases. From a strictlyscientific perspective, hESCs representa unique and powerful model system tostudy human development at the cellular level. For example, hESC experimentationwill allow us to monitor the molecular,biochemical and embryological underpin-nings of human development and disease.From a clinical perspective, hESCs mayserve as a viable treatment option against

various diseases in which cells need to bereplaced, repaired, or replenished. It hasbeen shown that hESCs are capable of pro-ducing the raw materials necessary for cellreplacement applications. From a phar-maceutical perspective, these pluripotentstem cells represent another arrow in thequiver with which to probe the safety andtoxicity of novel drugs. As a result, hESCsprovide a means for accelerating the timeit takes to develop and approve a noveldrug product.

SOURCES OFPLURIPOTENT CELLSPluripotency refers to the ability of a cellto produce all cell types of a mature organ-

ism. In embryological terms, pluripotencyrefers to the ability of cells to producederivatives of all three major germ lay-ers — ecto-, endo- and meso-derm. Thefirst pluripotent cell types to be isolatedand recognized to exhibit several proper-ties associated with embryonic cells wereembryonal carcinoma cells (ECCs), whichcan be derived from germ cell tumors andembryonic germ cells (EGCs), in vitro .

However, the nature of origin for EGCsand ECCs renders them unsuitable for therapeutic applications. The derivation of mouse embryonic stem cells in 1981 2,3 wasinstrumental in the development of reversegenetic approaches in mice (e.g. knock-out and knock-in technologies) and in thesubsequent isolation of hESCs. Hamperedby political, ethical and societal concerns,progress in hESC research was slow. Inresponse to federal funding restrictions onhESC research, several states, most nota-bly California, made available additionalfunding sources specifically for hESCresearch. To date, the debate surroundingthe field of hESC research is as lively asever. It is argued that the most dramaticd e v e l o p m e n tin the studyof pluripo-tent stem cellsoccurred in

2006 whenTakahashi and

Y a m a n a k apresented anelegant (andheroic) set of experiments inwhich differen-tiated cells werere-programmedto a cell typewith all theproperties of ESCs, a so-called inducedpluripotent stem cell (iPSC) state 4. Throughthe introduction of a handful of transcrip-tion factors (including Oct4, Nanog, Sox2,Klf4, Lin28 and Myc), the epigenome of a differentiated cell type can be re-set toan embryonic-like state. Recently, signifi-cant advances have been made in improv-ing this technology, such as the applica-tion of a canonical Wnt protein in place of the Myc oncogene 5, the reprogramming of terminally differentiated B lymphocytes 6,the use of non-integrating adenoviral vec-tors to introduce the reprogramming fac-

tors 7, and the use of recombinant proteinsonly 8. While iPSC technology requiresfurther research and development beforesuch cells would be suitable for clinicalapplications, previous studies have madepossible the generation of a large number of pluripotent cells with varying geneticbackgrounds.

MAINTENANCE ANDEXPANSION OF hESCs

A tightly regulated transcription comprisedof Oct4, Sox2, Nanog and other transcrip-tion factors underlies the maintenance of an undifferentiated state of pluripotentcells. The undifferentiated state of hESCs

in long-termculture isattributed inlarge part totheir extra-

cellular envi-r o n m e n t .Undifferenti-ated humane m b r y o n i cstem cells aremost reliablym a i n t a i n e din co-culturewith feeder cells, suchas the com-

monly used mouse embryonic fibroblasts(MEFs). While the optimal compositions of extracellular matrices and growth mediumhave not been fully defined, several com-ponents, including FGF2, Activin A andFibronectin, have been shown to substan-tially improve hESC culture in the absenceof feeder cells. Research on the underlyingmechanisms maintaining pluripotency hasled to the concept of a “ground state”— acondition of cellular “self-sufficiency moreakin to that of uni-cellular organisms thanthe interdependence generally exhibitedby metazoan

Introduction toPluripotent Stem Cells

REVIEW IN REWIND 3

Mitochondria:Cellular Saviors or theBeginners of the EndMetabolic processes occurring inmitochondria play a critical role inmeeting the energy demands of cells...

RESEARCHER'S CORNER 5

Complement C5aReceptor ExtractionOne of the important informational needsfor designing drugs is to recognizethe specific interaction of...

PRODUCT CITATION 7

ELISA Kit EvaluationOur aim was to conduct an analytical

validation in a routine laboratorysetting of the cerb-B2/c-neu...

PRODUCT SPOTLIGHT 6

RapidStep ECLRapidStep™ ECL Reagent is a chemiluminescentsystem for detection of horseradishperoxidase (HRP) on Western blots...

TOPICS

"A major challenge

in this research arenais the developmentof fully defined andoptimized cultureconditions..."

...continued on page 2

EMD4BiosciencesNewsletter A Calbiochem® and Novagen®Product Brand Newsletter



2cells”9. A major chal-

lenge in this research arena is the development of fullydefined and optimized culture conditions that promoteand maintain the highly proliferative ground state char-acteristic of ESCs.

DIFFERENTIATION OFHESCS AND iPSCs As derivation and maintenance of hESCs and iPSCsbecome more straightforward, a major remaining obstacleis their differentiation into specific lineages that are suita-ble for transplantation into animal models and eventuallyhumans. Studies of early mouse embryogenesis and devel-opment have been critical in elucidating the steps thatlead from the early embryonic stages from which ESCs ar ederived to differentiated and mature cell types and tissues.For example, primitive streak (PS) formation requires Wntsignaling 10; in comparison, treatment of ESCs with Wnt3aappears to promote a mesendodermal fate 11,12 , the celltype specified during PS formation. Studies in which ESCsare differentiated into mature cell types reiterate the basicprinciples of early embryogenesis, and the generation

of various cell types and tissues can be readily detectedby expression of markers that are expressed during the

various embryonic stages. Such markers have also pr ovenuseful for the enrichment of cells of interest through cellsorting technologies.

Protocols developed to date rely on available tools andreagents to coerce ESCs along distinct stages that mimic

the various developmental stages. For example, genera-tion of cell clusters analogous to Islets of Langerhans, thefunctional units in the pancreas that monitor blood sugar levels and produce insulin, involves a 2 to 3 week regimenof culture manipulations. Such modifications start withthe initial formation of a definitive endoderm (by high

Activin A/Wnt exposure) followed by the coordinatedstimulation and inhibition of signaling pathways, includ-ing FGF and Hedgehog signaling 13 . Generation of neuro-nal populations from human embryonic stem cells can beachieved through isolation of neural progenitors and their subsequent treatment with growth factors, such as FGFand SHH 14 . Recently, cells with hematopoietic stem cellproperties were isolated from human embryonic stem cellsby the co-culture with cells from hematopoietic niches 15 .In the future, it will be cr itical to identify the exact cock-tail of factors that drive pluripotent ESCs or iPSCs into

specific mature cell types.

CONCLUSION While the potential applications of pluripotent cells, either hESCs or iPSCs, are immense, many challenges remainbefore these cells can be exploited to their full potential.

As basic science surges ahead in elucidating the mecha-nisms that drive human development using pluripotent

cell types, close partnerships between academic centers,the biotechnology sector and big pharma are critical torealize the full potential of this exciting technology.REFERENCES

1. Thomson, J. A., et. al. (1998).Science 282 , 1145-1147.2. Evans, M. J., and Kaufman, M. H. (1981).Nature 292 , 141-156.3. Martin, G. R. (1981).PNAS 78 , 7634-7638.4. Takahashi, K., and Yamanaka, S. (2006).Cell 126 , 663-6765. Marson, A., etl. al. (2008).Cell Stem Cell 3 , 132-135.6. Hanna, J., et. al. (2008).Cell 133 , 250-264.7. Stadtfeld, M., et. al. (2008).Science . 322 , 945-9.8. Zhou, H., et al. (2009).Cell Stem Cell 4, 381-384.9. Ying, Q. L., et. al. (2008).Nature 453 , 519-523.10. Liu, P., et. al. (1999).Nat Genet 22 , 361-365.11. Bakre, M. M., et. al. (2007).J Biol Chem 282 , 31703-31712.12. Ten Berge, D., et. al. (2008).Cell Stem Cell 3, 508-518.13. D'Amour, K. A., et. al. (2006).Nat Biotechnol 24 , 1392-1401.14. Lee, H., et. al. (2007).Stem Cells 25 , 1931-1939.15. Ledran, M. H., et al. (2008).Cell Stem Cell 3 , 85-98.

continued from cover story...

We offer a range of products for stem cell researchBelow are some examples of products offered

For a complete product listing or more information visit:

Product Highlights

Small MoleculeRegulators

Product Name Brief Description

GSK-3 Inhibitor IX (Cat. No. 361550) Activates the Wnt-signaling pathway and sustains pluripotency in human and murine ESCs (embryonic stem cells).

GSK-3ß Inhibitor XII, TWS119 (Cat. No. 361554) Selectively induces neuronal differentiation in both pluripotent murine embryonal carcinoma cells and embryonic stem cells.

Stem Cell Proliferation Inhibitor (Cat. No. 569620) Acts as a natural inhibitor of pluripotent hematopoietic stem cell proliferation.

U0126 (Cat. No. 662005) Acts as an immunosuppressant by effectively blocking IL-2 synthesis and T cell proliferationwithout affecting the long-term outcomes of either T cell activation or tolerance.

Y-27632 (Cat. No. 688000) Prevents apoptosis and enhances the survival and c loning efficiency of dissocia ted ESCs without affect ing their plur ipotency.

Growth Factorsand Cytokines


rhFGF-2 ( Cat. No. 341595) Stimulates proliferation of a wide variety of stem cells. Potent mitogen for bone cells.

rhSCF (Cat. No. 569600) Hematopoietic growth factor that stimulates the growth of cells of multiple lineages.

hTGF-1 (Cat. No. 616450) Promot es apoptosis in resting human B lymphocytes, glioma cells, and trigeminal neurinomal cells.

Lineage MarkerAntibodies


Nanog pAb (Cat. No. SC1000) Nanog directs propagat ion of undifferent ia ted ESCs. I t i s res tr ic ted to founder cells f rom which ESCs can be der ived.

Sox2 mAb (245610) (Cat. No. SC1002) Sox2 plays a role in specifying the f irst three l ineages that are present a t egg implantation.

Notch 1 mAb (8G10) (Cat. No. 491010) Notch 1 is a transmembrane protein involved in the development and determination of cell-fate.

Related Antibodiesand Kits


APC (Ab-7) mAb (CC-1) (Cat. No. OP80) APC modulates ESC different ia tion through the Wnt signal ing pathway.

c-Myc (Ab-1) mAb (9E10) (Cat. No. OP10) Amplification of thec-myc gene has been found in several types of human tumors and is a key regulator of stem cell renewal.

VE GF EL IS A K it (C at . N o. QIA 51 ) Qu ant ifi es VE GF lev el s i n h uma n c el l cul tu re sup er na ta nt s, ser um, an d p las ma sam pl es .

TGF- ELISA Kit (Cat. No. QIA61) Quantifies TGF- in human serum, cell lysates, tissue culture supernatants, and plasma samples.

Accessory ProductsProduct Name Brief Description

Fibronectin, Human Plasma (Cat. No. 341635) Native fibronectin purified from human plasma. Effective agent for promoting attachment of cells to commonly used culture substrates.

FluorPreserve™ Reagent (Cat. No. 345787) A water-soluble, non-fluorescent mounting medium that provides a semi-permanent seal for long-term storage of slidepreparations. For use with Fluorescein, Rhodamine, Texas Red, Cy2™, Cy3®, Cy5®, Phycoerythrin, and Allophycocyanin.

G 418 Sulfa te (Cat. No. 345810) Widely used in the selection of eukaryotic express ion vectors carrying the bacter ia l neoR/kanR genes.

L-Glutamine (Cat. No. 3520) An unstable essential component in cell culture applications.

Product Name Size Cat. No.

NEW StemSelect™ Small Molecule Regulators 384-Well Library I 1 ea 569744

www.emdbiosciences.com/StemCells

Cover Story | EMD4Biosciences Newsletter | July 2010



3Chandra Mohan, Ph.D. EMD Chemicals, San Diego, CA 92121

Metabolic processes occurring in mitochondria play a criticalrole in meeting the energy demands of cells. However,eukaryotic cells have also evolved to possess the capabilityto rupture this indispensable organelle and assemble a groupof proteins to bring about the rapid destruction of the cell.Until the last decade, the role of mitochondria in apoptosiswas rather obscure. This was due to the fact that mitochondriado not exhibit any prominent morphological changes duringapoptosis. They contain all the components necessaryto annihilate the cell by apoptosis, without giving anyappearance of self-destruction. However, studies have shownthat mitochondria undergo several major changes, includingthe changes in their membrane integrity, even before anyclassical signs of apoptosis appear. Susin, et al. (1998)divided the apoptosis phenomenon into three phases - a pre-mitochondrial phase, which involves the activa tion of damage

pathways; a mitochondrial phase, which involves the loss of mitochondrial functions; and a post-mitochondrial phase,in which proteins released from the mitochondria activatecaspases and nucleases to facilitate the ultimate cell's demise.

Bax and other pro-apoptotic members of the Bcl-2 familyshow some structural similarities with pore-forming proteins.Hence, it is believed that Bax can form transmembrane pores

across the outer mitochondrial membrane, which leads toa loss of membrane potential. The localization of Bax hasbeen shown to change from the cytosol to the mitochondriaduring apoptosis. Bcl-2 and other anti-apoptotic membersof the Bcl-2 family are located in the outer mitochondrialmembrane. Bcl-2 is especially enriched at contact sites wherethe inner and outer membranes come in close proximity. Itacts on mitochondria to counteract the action of pore-formingBax protein.

A number of mechanisms have been described that highlightthe mitochondrial involvement in apoptosis. Under normalconditions, the mitochondrial inner membrane is impermeableto all but a few selected metabolites and ions. This is essentialto maintain the membrane potential and pH gradient that drive

ATP synthesis through oxidative phosphorylation. High levels

of cytosolic Ca 2+ and excessive concentrations of reactive

oxygen species are known to contribute to the opening of the mitochondrial permeability transition pore (PTP), whichdepolarizes the mitochondria. The PTP participates in theregulation of matrix Ca 2+ levels, pH, and volume. Adeninenucleotide translocator (ANT) and the voltage-dependentanion channel (VDAC) are the two principal componentsof PTP. The PTP operates at the inner and outer membranecontact sites and creates a channel that allows non-specificpassage of ions and molecules smaller than 1.5 kDa. Theopening of this channel in the inner membrane allows for equilibration of ions within the matrix and the intermembranespace. This dissipates the electrochemical gradient ( ∆ψ m) anduncouples the respiratory chain, leading to the cessation of

ATP production. The disruption of the ∆ψ m is attributed to belargely responsible for the damage of the inner mitochondrialmembrane. This event is believed to occur even before DNAfragmentation indicating that mitochondrial injury is an early

event in apoptosis. The use of cationic lipophilic dyes, such asDiOC6(3) has shown that a disruption of the ∆ψ m may evenprecede the activation of caspases. Several apoptosis-inducingagents are known to trigger mitochondrial uncoupling thatresult in a diminution of ∆ψ m.

In the low-conductance state, the opening of PTP is pHdependent, which permits the diffusion of only small ions and

then closes spontaneously. However, in the high-conductancestate, the channel is stabilized in an open position and allowswater and larger molecules to enter the protein-rich matrix,which results in mitochondrial swelling. Consequently, theinner membrane, which has a far greater surface area thanthe outer membrane, unfolds, exerting pressure on the outer membrane. As a result, the outer membrane ruptures andcauses the release of pro-apoptotic factors, such as apoptosis-inducing factor (AIF) and Cyt c into the cytosol where Cytc and apoptosis protease activating factor-1 (Apaf-1) act ascohorts in the activation of caspase-9.

Cyt c, Apaf-1, ATP, and pro-caspase-9 come together toform a complex known as the apoptosome. Binding to Cyt cmakes Apaf-1 more competent at binding pro-caspase-9. TheN-terminus of Apaf-1 that contains the caspase recruitment

domain (CARD), which is shared by several caspases. Since

Apaf-1 does not have any caspase activity, pro-caspase-9is believed to be activated through autocatalysis in theapoptosome. Thus, Cyt c release from the mitochondria isconsidered a key signal that initiates the irreversible eventsin cell death.

The AIF, a 67 kDa nuclear encoded flavoprotein, is generallyconfined to the mitochondrial intermembrane space. AIF hasbeen termed a “fellow escapee” by Waterhouse and Green(1999) that ensures eventual cell death. AIF is capable of activating caspase-3 and endonucleases independently of Cyt c. It induces large-scale DNA fragmentation and a lossof mitochondrial transmembrane potential. A wide range of chemotherapeutic agents are known to induce AIF releasefrom the mitochondria. On the other hand, anti-apoptoticagents, such as Bcl-2, inhibit the translocation of AIF. Another mitochondrial factor, second mitochondrial activator of

caspases/direct inhibitor of apoptosis binding protein with lowpI (Smac/DIABLO), has been identified that acts by binding tothe inhibitor of apoptosis proteins (IAPs), thereby potentiatingapoptosis by relieving the inhibition of IAPs on caspases.Smac is an intermembrane protein that is released frommitochondria along with Cyt c and promotes Cyt c-dependentcaspase activation. WOX1, a mitochondrial oxidoreductase,also migrates to the nucleus following induction of apoptosis.

Phosphorylation of WOX1 at Tyr 33 is considered to be essentialfor its cytotoxic effects in the nucleus. Endonuclease G, a 50kDa nonspecific DNA/RNA nuclease, is also released frommitochondria and translocates to the nucleus during apoptosisand contributes to chromatin fragmentation.

Despite volumes of data linking mitochondrial events toapoptotic cell death, some researchers believe that the r eleaseof Cyt c is only distal to caspase activation in Fas-activatedapoptosis, and the mitochondrial signaling may not beessential for apoptosis. In their opinion, primary apoptosissignals are routed directly to caspase activation, andmitochondrial events only contribute to the amplification of cell death. These conclusions may be based on the observationthat caspase inhibitors, such as Z-VAD-FMK, block therelease of Cyt c from mitochondria. It is of interest to mention

Mitochondria: Cellular Saviors or the Beginners of the End

MitochondrialRole in ApoptoticCell Death

EMD4Biosciences Newsletter | July 2010 |Review in Rewind



4that both caspase-8, activated via the receptor-mediatedpathway, and caspase-9, activated via the mitochondrialpathway, participate independently in the activation of pro-caspase-3, the active formof which is termed the “centralexecutioner of apoptosis.” A

point of clinical considerationmay be the selective eradicationof transformed cells by highlyselective mitochondrial-specificchemotherapeutic agents,which may either prevent thedecrease in ∆ψ m or block theBcl-2-mediated stabilization of mitochondrial membranes.

Some of the factors that mediateDNA damage in apoptosis areproteins that transmit apoptoticsignals out of the nucleus andexpedite apoptotic cell death. Nur77/TR3 is recognizedas a nuclear messenger of apoptosis that translocates tothe mitochondria and releases Cyt c. It binds to Bcl-2 and

triggers a conformational change, which exposes the pro-apoptotic BH3 domain. This converts Bcl-2 from an anti-

apoptotic to a pro-apoptotic factor. Nur77/TR3 contains twonuclear localization and three nuclear export sequences. Itis shown to heterodimerize with retinoid X receptor α and

is actively transported from thenucleus to the mitochondria incells undergoing apoptosis.

Research over the last few years has shown that salthoughcaspases are vital for the typicalapoptotic morphology, caspase-independent cell death alsoco-exists with caspase-dependentdeath. It is shown that inhibitionof caspase activities in Jurkat cellscan still lead to cell death by acaspase-independent mechanism.However, cells prefer caspase-dependent apoptosis over caspase-independent cell death. The latter

death process creates a huge oxidative stress load on the celland may even cause damage to the nearby cells.

Defects in p53 and bcl2 genes are associated with proliferativedisorders and apoptosis. It's believed that p53, a key tumor-

suppresser protein, accumulates when DNA is damaged andarrests the cell cycle at the G1 phase to allow extra timefor repairs. However, if the repair process fails, p53 triggersapoptosis. When p53 is dysfunctional, apoptosis fails tooccur. Also, an over-expression of Bcl-2 retards the normalapoptotic process. Anticancer drugs mediate their effect by

triggering apoptosis. There is strong evidence to suggestthat cancer cells can increase their resistance to anticancer drugs by increasing the expression of Bcl-2 that leads toinhibition of apoptosis. Inactivation of p53 also contributesto the initiation and progression of cancer. Hence, p53 statusbecomes a strong determinant of response to treatmentwith anticancer agents. Genotoxic compounds such as5-fluorouracil, etoposide, and adriamycin can increasep53 levels and cause growth inhibition in tumor cells. Themitochondrial (intrinsic) apoptotic pathway, activated bycytotoxic drugs ultimately leads to the activation of caspasesthat cause cell death in tumor cells. In addition, activationof the receptor-linked (extrinsic) apoptotic pathway leadsto enhanced sensitivity of tumor cells to cytotoxic agents.Hence, apoptosis is an important area of study where theability to achieve a significant therapeutic index anddifferentiating normal cells from tumor cells may lower the

threshold at which cell injury triggers apoptosis.

Cyt c release fromthe mitochondria isconsidered as a keysignal that initiatesthe irreversibleevents in cell death

REFERENCES:

Klee, M., et al. 2009.EMBO J. 28 , 1757.Ow, Y.L., et al. 2008.Nat. Rev. Mol. Cell Biol. 9, 532.Salvesen, G.S., and Riedl, S.J. 2008.Adv. Exp. Med. Biol. 615 ,13.Belizario, J.E., et al. 2007.Braz. J. Med. Biol. Res. 40 , 1011.Ardehali, H. 2005.J. Bioenerg. Biomembr. 37, 171.Broker, L.e., et al. 2005.Clin. Cancer Res. 11, 3155.Ferrando-May, A. 2005.Cell Death Differen. 12, 1263.Green, D.R. 2005.Cell 121, 579.Hail. N. Jr. 2005.Apoptosis 10, 687.Orrenius, S., et al. 2004.Toxicol. Lett. 149 , 19.Philchenkov, A. 2004.J. Cell. Mol. Med. 8, 432.Zhang, A., et al. 2004.Neuroembrology 3, 47.Liston, P., et al. 2003.Oncogene 22 , 8568.

Mayer, B., and Oberbauer, R. 2003.New Physiol. Sci. 18, 89.Ni, C-Z., et al. 2003.J. Mol. Recog. 3, 121.Finkel, E. 2001.Science 291 , 624.Roberts, D.L., et al. 2001.J. Cell Biol. 153 , 221.Shi, Y. 2001.Nat. Struct. Biol. 8, 394.Li, L.Y., et al. 2001.Nature 412, 95.Daugas, E., et al. 2000.FEBS Lett. 476 , 118Gorman, A.M., et al. 2000.Dev. Neurosci. 22 , 348.Gottlieb, R.A. 2000.FEBS Lett. 482 , 6.Shimizu, S., et al. 2000.J. Biol. Chem. 275 , 12321.Srinivasula, S.M., et al. 2000.J. Biol. Chem. 275 , 36152Crompton, M. 1999.Biochem. J. 341, 233.Susin, S.A., et al. 1999.Nature 397, 441.Waterhouse, N.J., and Green, D.R. 1999.J. Clin. Immunol. 19, 378.

Cai, J., et al. 1998.Biochim. Biophys. Acta 1366 , 139.Reed, J.C., et al. 1998.Biochim. Biophys. Acta 1366 , 127.Susin S.A., et al. 1998.Biochim. Biophys. Acta 1366 , 151.Knudson, C.M., and Korsmeyer, S.J. 1997.Nat. Gen. 16, 358.Kroemer, G., et al. 1997.Immunol. Today 18 , 44.Nagata, S. 1997.Cell 88 , 355.

Yin, C., et al . 1997.Nature 385 , 637.Zhivotovsky, B., et al. 1997.Arch. Biochem. Biophys. 230 , 481.Cuvillier, O., et al. 1996.Nature 381 , 800.Hsu, H., et al. 1996.Cell 84 , 299.Liu, X.S., et al. 1996.Cell 86 , 147.Susin, S.A., et al. 1996.J. Exp. Med. 184 , 1331.Wertz., I.E., and Hanley, M.R. 1996.Trends Biochem. Sci. 21, 359.Whyte, M., et al. 1996.Trends Cell Biol. 6 , 245.

Zamzami, N., et al. 1996.J. Exp. Med. 183 , 1533.Cifone, M.G., et al. 1995.EMBO J. 14, 5859.Carston, D.A., and Lois, A. 1995.Lancet 346 , 1009.Enari, M., et al. 1995.Nature 375 , 78.Kischkel, F.C., et al. 1995.EMBO J. 14, 5579.Hannun, Y.A., et al. 1994.J. Biol. Chem. 269 , 3125.Reed, J.C. 1994.J. Cell Biol. 124 , 1.Smith, C.A. et al. 1994.Cell 76 , 959.Wilson, K.P., et al. 1994.Nature 370 , 270.Fisher, T.C., et al. 1993.Cancer Res. 53 , 3321.Cohen, J.J., and Duke, R.C.. 1992.Annu. Rev. Immunol. 10 , 267.Lane, D.P. 1992.Nature 358 , 15.Thornberry, N.A., et al. 1992.Nature 356 , 768.

Related Kits

Related Antibodies and Proteins

Related Inhibitors and Biochemicals

Related Dyes and Stains

Detection of PDH-E1a at Ser293 by immunocytochemistry: Samples: COS7 cells were incubated in the presence and absence of 5 mM dichloroacetate (DCA) for 2 hrs. Cells were incubated with 100 nM MitoTracker® Red to visualize mitochondria(red) for 20 minutes before fixation with 3.7% formaldehyde, followed by a3 min fixation with methanol at -20 degrees C, and permeabilization in PBS containing 0.1% Tween 20, 0.3% TritonX-100, and 6% BSA. Cells were thenincubated with either PhosphoDetect Anti PDH-E1a Ser293 Rabbit pAb (Cat.No. AP1062) at 500ng/mL (green) or a pan specific PDH-E1a antibody (green).The secondary antibody used was anti-Rabbit IgG (Goat) Alexa Fluor®488 Conjugate. Nuclei were stained with 300 nM DAPI (blue) for 1 min before viewing. Data courtesy of Matthew Rardin, University of California San Diego.

mitochondria Phospho PDH merge

merge

0 h r

2 h r

D C A

2 h r

D C A

mitochondria PDH

MitoTracker® and Alex Fluor® are trademarks of Life Technologies.Triton is trademark of The Dow Chemical Company.

Product Application Sample Type Number of Tests/Samples Cat. No.

InnoCyte™ Flow Cytometric Cytochrome c Release Kit FC, IF Intact Cells 50 Cell stainings CBA077

Cytochrome c ELISA Kit ELISA Cell lysates 96 Tests QIA74

Cytochrome c Release Apoptosis Assay Kit IB Cell Extracts 100 Tests QIA87

MitoCapture™ Apoptosis Detection Kit FC, IF Cell suspension oradherent cells

100 Tests 475866

ProteoExtract® Cytosol/Mitochondria Fractionation Kit Cells 100 Extractions QIA88

Hsp27 ELISA Kit ELISA CL, P, S, T 96 Tests QIA119

Cu/Zn Superoxide Dismutase ELISA Kit ELISA CS, P, S, AF 96 Tests QIA97

Product Application Species Reactivity Cat. No.

Anti-Cyclophilin D Mouse mAb (E11AE12BD4) IB, IC bovine, human, rat AP1035

Anti-Cytochromec (Ab-1) Sheep pAb IB, IF, IP canine, human, rabbit, rat PC323

Anti-Cytochromec Mouse mAb (6H2.B4) FC, IC, IP, NOT IB human, mouse, rat AP1030

Anti-Cytochromec Mouse mAb (7H8.2C12) FC, IB, IC, NOT IP, PS avian, horse, human,mouse, rat

AP1029

Cytochromec , Equine Heart N/A N/A 250600

Anti-F1F0-α Mouse mAb (7H10BD4F9) CIA, IB, IC bovine, human, mouse, rat AP1036

Anti-F1F0-b Mouse mAb (3D5AB1) CIA, IB, IC, IP bovine, human, mouse, rat AP1037

Product Purity Solubility Cat. No.

Atractyloside, Dipotassium Salt,Atractylis gummifera ≥ 95% TLC H2O 189300

Bongkrekic Acid, Triammonium Salt ≥ 92% HPLC 2N NH4OH or EtOH 203671

Carbonyl Cyanide m-Chlorophenylhydrazone ≥ 95% HPLC DMSO or EtOH 215911

Mitochondrial Permeability Transition Pore Reagents Set Multiple Multiple 475876

Oligomycin ≥ 90% HPLC EtOH 495455

Rotenone ≥ 98% TLC CHCl3, DMSO, or EtOH 557368

Ru360 ≥ 97% Electro-phoresis

Deoxy. H2O 557440

Smac-N7 Peptide ≥ 95% HPLC DMSO or H2O 567370

Smac-N7 Peptide, Cell-Permeable ≥ 95% HPLC DMSO 567375

Valinomycin,Streptomyces fulvissimus ≥ 93% HPLC Multiple 676377

Product Purity Solubility Cat. No.

3,3'-Dihexyloxacarbocyanine Iodide ≥ 98% HPLC EtOH 305110

JC-1 ≥ 95% HPLC DMF or DMSO 420200

Rhodamine 123 ≥ 98% UV DMSO or MeOH 555505

Applications: IB - Immunoblot, FC - Flow cytometry, IF - Immuno uorescence, IP - Immunoprecipitation, PS - Paraf n sections, CIA - Cell inhibition assay Sample Type: AF - Amniotic uid, CL – Cell Lysates, CM – Culture Medium, R – Recombinant protein, S – Serum, P – Plasma, T – Tissue

Review in Rewind| EMD4Biosciences Newsletter | July 2010



5Complement C5a Receptor ExtractionExtraction of complement C5a receptor from the lipid raft on HMC-1 cells by a combinational useof ProteoExtract® Transmembrane Protein Extraction Kit (TM-PEK) and methyl-ß-cyclodextrin

C5a receptor: 43 kDa

α-C5aR rabbit IgG

CholeratoxinB: 32 kDa

α-CholeratoxinB goat IgG

GM-1/PE C5aR/FITC

© 2007 Immobilon® is a registered trademark of Millipore Corporation.© 2009 FluoView™ is a trademark of Olympus Corporation. ©2010 ECL Plus™ is a trademark of GE Healthcare companies

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

Figure 1: Lane 1, 2, and 3 denote samples of cytoplasm, plasma membrane, and other fraction proteins separated by using M ßCD(-)/TM-PEK Regent B, respectively. Lane 4, 5, and 6 denoted samples of cytoplasm. plasma membrane, and other fraction proteins separated by using M ßCD(+)/TM-PEK Regent B, respectively. Lane 7-12 denoted similar samples, but using TM-PEK Regent Bx2.

Figure 2: C5a receptor (CD88) and GM-1 on RAFT fraction of HMC-1 cells were visualized by FITC-conjugated anti-CD88 rabbit IgG and PE-conjugated anti-CholeratoxinB goat IgG, respectively.

Figure 1 Figure 2

Hiroshi NishiuraDepartment of Molecular Pathology, Faculty of Medical and Pharmaceutical Sciences,Kumamoto University, Kumamoto, Japan.

ABSTRACTOne of the important informational needs for designing drugs isto recognize the specific interaction of 7-transmembrane-type Gprotein-coupled receptors (GPCRs) with their ligands. However,it still is very difficult to extract GPCRs from the cholesterol/sphingolipid micro-domain, lipid raft. Recently, methyl-ß-cyclodextrin (MßCD) and TM-PEK were reported to support theextraction of proteins from the lipid raft. To examine the effectof MßCD on the extraction of C5aR, which is one of the well-known GPCRs on the lipid raft, by TM-PEK, we first confirmedthe localization of the C5aR in monosialotetrahexosylganglioside(GM-1)-containing raft of the human mast cell line, HMC-1, byconfocal laser scanning microscopy (CLSM) analysis. An efficientextraction of both C5aR and GM-1 from HMC-1 was possibleonly when using TM-PEK Reagent B undiluted with ExtractionBuffer 2 (HMC-1 cells were pretreated with 10 µM MßCD).The detection of C5aR and GM-1 was performed by westernblotting analysis. Other combinational uses of the reagents didnot improve the extraction. MßCD seems to somewhat supportthe extraction of GPCRs from the lipid raft of the cells by usingTM-PEK.

INTRODUCTIONGPCRs including C5aR are well recognized to be the targetsof many drugs 1. Although understanding the functionalsignificance of the wide structural diversity of C5aR is importantfor designing future therapeutic antagonists, details are notcompletely understood 2,3. In order to observe the structure of C5aR in detail, we needed to extract the functional C5aR from thedetergent-resistant lipid raft. Recently, MßCD and TM-PEK werereported to support such extraction 4. Therefore, we examinedthe effect of MßCD on extraction of the C5aR from HMC-1 byusing TM-PEK.

METHODSFor Fluorescent Immuno-Cytochemistry analysis of the cellsurface expression of C5aR and GM-1, cells were stainedwith FITC-conjugated anti-C5aR mouse IgG 1 (Santa CruzBiotechnology Inc., CA) and PE-conjugated anti-Choleratoxin Bgoat IgG for 40 min at 4°C. Cells were observed by an OlympusFluoroview™ FV300 microscope (CLSM) (Olympus Corporation,Tokyo, Japan).

SDS-PAGE and Western Blot- HMC-1 was kindly gifted by Dr. J.H. Butterfield of Mayo Clinic, USA. The cells (2 x 10 6 cells) wereincubated in 500 µl of phosphate-buffered saline (PBS, pH 7.3)with or without 10 µM MßCD for 30 minutes at 22°C to removecholesterol. After washing 3 times in 1 ml of cold PBS, cellswere treated in 500 µl TM-PEK Extraction Buffer 1 containingProtease Inhibitor Cocktail SET III from TM-PEK (Merck KGaA,Tokyo, Japan) for 10 minutes at 4°C. Membrane and cytoplasmicproteins were separated into pellet and supernatant fractions,respectively by centrifuging at x1,000 g. Membrane proteinswere incubated for 45 minutes at 22°C in four different reagents:1) 100 µl of TM-PEK Reagent A, 2) 100ul of TM-PEK Reagent A(two times-diluted with Extraction Buffer 2, 3) 100 µl of TM-PEKReagent B and 4) 100ul of TM-PEK Reagent B (two times-dilutedwith Extraction Buffer 2. After centrifuging at x 15,000 g,proteins in supernatants and pellets were named as membrane-extracted proteins and membrane-bound proteins, respectively.Cytoplasmic proteins, membrane-extracted proteins, andmembrane-bound proteins were precipitated in 80% acetone,and each precipitant was re-suspended in 100 µl of SDS-loadingbuffer. 10µl of sample were loaded on 12% SDS-polyacrylamidegel, and then proteins were transferred to an Immobilon®-P SQ

membrane (Millipore Inc., Billerica, MA) 5. After treating with 1%Block Ace™ for 1 hour at 22°C, the rabbit or goat IgG was usedas primary antibody. Incubation was performed for 1 hour at22°C. HRP-conjugated anti-rabbit IgG goat IgG or anti-goat IgGsheep IgG was used as secondary antibody and incubation wasperformed for 30 minutes at 22°C. The HRP was detected usingthe ECL Plus™ Western blot detection system (GE Healthcarecompanies, Piscataway, NJ).

RESULTS AND DISCUSSIONSBefore examining the effect of MßCD on extraction, the presenceof the C5aR on lipid raft of HMC-1 was confirmed by fluorescentimmuno-cytochemistry. As shown in Fig. 1, almost all of theFITC-labeled C5aR was observed as a yellow spot by fusionwith PE-labeled GM-1-containing raft in CLSM analysis. Thisindicates that the C5aR is localized in the lipid raft of HMC-1.To examine the effect of MßCD on the C5aR extraction, HMC-1pretreated with or without MßCD was incubated in undiluted or two times diluted TM-PEK Reagent B, respectively (Fig. 2). Thelargest amount of the C5aR recovery and the detection of GM-1by western blot was enabled by the combinational use of MßCDand undiluted TM-PEK Reagent B. By single use of the solutionsor combinational use of MßCD and TM-PEK Reagent A, C5aRwas not extracted (data not shown). From above data, 10 µMMßCD supports the extraction of the C5aR from the lipid raft of HMC-1 by using TM-PEK. We have to continuously examine themost efficient condition to extract not only C5aR but also other GPCRs from other types of cells.

REFERENCES

1. Lagerström, M.C. and Schiöth, H.B. 2008.Nature Rev. Drug Disc. 7, 339.2. Monk, P.N., et al. 2007.Br. J. Pharm. 152 , 429.3. Yamamoto, T. 2007.Pathol. Int. 57, 1.4. DiPilato, L.M. and Zhang, J. 2009.Mol. Biosyst. 5, 832.5. Kyhse-Andersen, J. 1984.J. Biochem. Biophys. Meth. 10 , 203.

Product Name Size Cat. No.

ProteoExtract® Transmembrane Protein Extraction Kit 1 Kit 71772

Protease Inhibitor Cocktail Set III, EDTA-Free(1 set is 5 x 1 ml)

1ml1 set 539134

Protease inhibitor Cocktail Set III, Animal-Free(1 set is 5 x 1 ml)

1ml1 set 535140

SignalBoost ™ Immunoreaction Enhancer Kit 1 Kit 407207

RapidStep™ ECL Reagent 100 ml 345818

For more information on the wide range of available Immuno-CytoChemistry products and protease inhibitors, please visit our website by referring to the following links:

www.emdbiosciences.com/westernblot andwww.emdbiosciences.com/proteaseinhibitors

EMD4Biosciences Newsletter | July 2010 |Researcher's Corner



6INTRODUCTIONRapidStep™ ECL Reagent is a chemiluminescent system for detection of horseradish peroxidase (HRP) on Western blots.This reagent provides a chemiluminescent signal that is bothintense and long-lasting. The unique feature that differentiatesthis system from other chemiluminescent systems is thatthe substrate and enhancer solution are premixed in ahomogenous, stable solution. A specially designed spraybottle, with a precision misting mechanism, is used to applythe solution directly onto the membrane.

METHODSThe best feature of RapidStep™ ECL is the convenience andease of use it provides since no pipeting or mixing is required.

After the last wash step in the Western blotting protocol, sprayRapidStep™ onto the membrane, just sufficient to ensure evencoverage. Usually this requires between 2 and 5 sprays for amini-gel precut membrane. Wetting the membrane more thannecessary does not provide any additional advantages; it isadvised to spray until the membrane is just moist. Incubatethe substrate on the membrane for 1-2 minutes. At this point,remove the excess liquid by touching the membrane againstabsorbent paper and enclose the membrane in a sheet of plasticwrap, being careful to remove any air bubbles between themembrane and the wrap. Place the wrapped membrane in afilm cassette, protein side up and expose to X-ray film for

varying amounts of time. Generally expose the membrane for several seconds and 1, 3, 5, and 10 minutes.

In order to obtain low background and crisp signals,optimization must be done with the primary and secondaryantibody. Optimizing these variables will reduce non-specificreactions and give strong specific results with an optimalsignal-to-background ratio. Researchers have noted thatsince RapidStep™ is more sensitive than other ECL reagents,it is best to reduce the amount of primary antibody. If youare currently using another ECL system, it will be necessaryto adjust experimental conditions with RapidStep™. Onceexperimental conditions are optimized, however, RapidStep™delivers superior results.

We have performed experiments in which RapidStep™ wascompared with Pierce and GE ECL substrates. The sensitivityof RapidStep™ is in the low pictogram range with minimalbackground interference.

CONCLUSIONIn summary, RapidStep™ allows for convenience and time-savings, without sacrificing quality. This reagent is versatilefor use with nitrocellulose as well as PVDF membranes. Inaddition, RapidStep™ is highly robust, where light emissioncan be detected up to 2 hours after addition to the membrane.

RapidStep™ ECL ReagentOne-step, easy to use, chemiluminescent substrate

RapidStep ™ECL is a one-step chemiluminescent substrate r eagent for use with horseradish peroxidase (HRP) labeled antibodies. This reagentis a convenient, cost-effective, and ready-to-use reagent that provides asuperior, longer-lasting signal than traditional ECL reagents. RapidStep™contains luminol and enhancer combined in one solution and is ready-to-use without mixing. A spray bottle is used to apply the solution directlyonto the membrane.

HRP

Primary antibody

Secondary antibody-HRP conjugate

lightHRP activates RapidStep™substrate, emitting light

2H2O2 + Luminol 3’-aminophthalate + N 2 + LightHRP

Convenient Just spray...that’s it! No need for mixing luminol and enhancer solution.No need to pipette or agitate membrane during substrate addition

Sensitive As little as 2 pg of protein can be detected

Long lasting Light emission is detected up to 2 hours after the addition of substrate

Versatile Can be used with either PVDF or nitrocellulose; compatible with film and CCD exposures

RapidStep™ provides superiorsensitivity as compared to otherenhancer reagents

Detection of recombinant Pit-1using decreasing amounts of protein

Sample: Recombinant Pit-1 protein

Primary Antibody: Anti-Pit-1 Rabbit pAb (1:2000 dilution)

500pg 250 50 10 2 500pg 250 50 10 2 500pg 250 50 10 2

RapidStep™ GE™ ECL™ Plus Pierce SuperSignal®

ECL™is a trademark of GE.SuperSignal® is a registered trademark of ThermoFisher Scientific.CDP-Star AP is a registered trademark of Perkin-Elmer

27 kDarPit-1protein

RapidStep™ECLA Convenient and Sensitive ECL Reagent

Product Name Size Comments Cat. No.

Rapi dStep™ ECL Reagent 100 ml Ultra-sensitive and convenient ECL; suf cient for2000 cm2 membrane area 345818

Related Products Size Comments Cat. No.AP Detection Reagent Kit 1 ea

5 eaComplete kit for chromogenic detection of alkalinephosphatase conjugates

69264-369264-4

AEC, hydrochloride 10 ml Precipitate peroxidase substrate for immunoblotting 152224CDP-Star ® AP Substrate 40 ml High sensitivity subst rate for al kali ne phosphat ase

conjugates69086-3

gLOCATOR™ Luminescent Labels 25 ea Labels to ensure unambiguous blot orientation 69102Development Folder 25 ea Transparent support for chemiluminescent exposures. 69137

SignalBoost™ Immunoreaction EnhancerKit

1 kit Ready-to-use signal enhancement reagent;suf cient for 2000 cm2 membrane area

407207

TrailMix™ Western Markers 25 lanesPrestained indicator proteins allow direct visualizationduring electrophoresis

70982-3

Rapidstain™ Reagent 1 l Enhanced Coomassie stain 553215

Product Spotlight | EMD4Biosciences Newsletter | July 2010



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