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RPPA applications present two common primary needs for users to obtain robust data…..

The first need is the required SENSITIVITY to detect low abundance proteins from LOWEXPRESSION LEVEL SAMPLES or from LABILE SAMPLES OF VERY SMALL MASS….

This has driven many users to adopt SIGNAL AMPLIFICATION methods to achievedetection of the pathways they seek to observe, with either Colorimetric or Fluorescentenhanced sensitivity techniques.

The SECOND user need for ROBUST RPPA data is DATA ACCURACY provided byCONFIDENT NORMALIZATION METHODS and BROAD DYNAMIC RANGE.

As we’ll see later, this ACCURACY is optimally provided by INTRA-SPOT MULTICOLORmultiplexed methods and this CANNOT BE PROVIDED by these current techniques usingamplification.

The ArrayCAM system presented here achieves BOTH the SENSITIVITY to detect lowabundance protein and provides ROBUST multi-color intra-spot data normalization.

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The Grace Bio-Labs ArrayCAM Proteomic Profiling System is a suite of thesecomplimentary components SPECIFICALLY DESIGNED and DEVELOPED to functionTOGETHER providing optimal assay performance

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Obtaining sensitive measurements of low abundance proteins begins with immobilizingas much protein on the array substrate as possible and Porous nitrocellulose provides thehighest protein binding capacity among microarray surfaces.

PNC non-covalently binds protein, retaining TERTIARY and QUATERNARY structurespreserving BINDING SITES and ANTIGENICITY.

This quantifies to 500X GREATER BINDING CAPACITY yielding HIGHEST S/N translatinginto 50X LOWER LOD’s.

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Porous Nitrocellulose is optimal when used in combination with Fluorescent detectionmodalities due to emission and excitation light RESONANT SCATTERING AMPLIFICATIONpropagated by the virtue of optimized pore size and structure harmonized towavelengths. This provides a 150 fold signal enhancement when compared to identicalquantities of bound probe to other surfaces.

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When employing fluorescent detection the inherent native autofluorescence of PNCmust be also considered.

At visible light emission wavelengths considerable auto-fluorescence is observed,however migrating detection wavelengths to the near IR range mitigates the presence ofautofluorescence.

In fact, moving from emission wavelengths of 635 nm to 800 nm, we observe a 4Xgreater S/N.

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To achieve optimal fluorescence in the near IR range with PNC, we employ a QuantumNanoparticle for assay detection.

The quantum nanoparticle (QNP) is a PROTEIN SIZED FLUORESCENT INORGANIC CRYSTALwhich is excited at a single wavelength while emitting very NARROW EMISSION bands,facilitating robust multi-color multiplexing.

40X Brighter than other organic fluorophores, these molecules are also PHOTO-STABLE,and won’t degrade or quench under REPEATED EXCITATION or due to other normalenvironmental conditions providing an ARCHIVABLE RECORD of assay data.

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The high binding capacity of PNC requires extremely effective blocking and requiresspecifically developed reagent sets for optimal results

As seen here, use of optimized blocking and assay reagents provides improved S/N andsensitivity. Super G blocks very strongly and can over-block for some applications.QBlock is a specially formulated non-protein blocker optimized for fluorescentapplications.

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RELIABLE IMAGING is a critical facet to confident RPPA results and is one of the mostEXPENSIVE COMPONENTS of RPPA assay systems.

Combining the unique SINGLE EXCITATION wavelength properties of QNC we havedeveloped a LOW COST MULTIWAVELENGTH ARRAY IMAGING INSTRUMENT whichprovides complete slide images in under 1 minute.

ArrayCAM contains four emission filters, and is typically equipped with 3 Near IR and oneColorimetric channel.

It is COMPACT and is offered in a BENCH-TOP or miniaturized version appendable torobotic high throughput liquid handling systems.

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To demonstrate the ArrayCAM imaging capabilities, we benchmarked against a GenePixconfocal laser scanner.

When observing slide dilution series imaged on both instruments, the ArrayCAMprovides identical signal-background intensities to that of the more expensive andcomplex GenePix confocal laser scanner.

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Finally, the ArrayCAM employs an extremely rapid easy to use automated software package toexpedite spot intensity measurements, eliminating manual alignment processes of typicalsoftware.

Here is a quick video to demonstrate the speed of the automated array image analysis,First you SELECT a TEMPLATE based on the commonly used GAL file.

Next, select the features you wish MEASURED and REPORTED as well as the background andintensity analysis methods.

OPEN the IMAGE

Then click to START the ANALYSIS

Here the software speeds through 640 spots in less than 1 minute, measuring, size, location,backgrounds and intensities

Then derived template can be confirmed, and adjusted if needed

Then the data is ready to review.

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The accuracy of the ArrayCAM automated spot finding software is compared here withthat of automated spot finding of the GenePix software. Both automated spot findingsystems are benchmarked against spot intensities obtained by manual spot location andanalysis on the same slide.

As seen HERE, Automated ArrayCAM software exhibited nearly perfect correlation to themanual benchmark method where the comparable Automated GenePix method, HEREdeviated greatly from the benchmark manual method.

The ArrayCAM results requires no additional manual template adjustments greatlyreducing image analysis time to obtain concordance with the correct spot intensities.

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This system as a whole enables us to reliably multiplex multiple detection colors withinan array without any cross-talk or signal bleed over.

In this example, we’ve probed cell lysates with three different species antibodies andthen simultaneously detected with three anti-species detection antibodies at wavelengthchannels, 800, 655 and 585 nm. Observing the control spots in the center of the arrays,we can see that there is essentially no cross reactivity or bleed over occurring betweenthese three detection channels.

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Application of intra-spot multiplexing provides increased assay throughput, and a veryrobust and reliable method of data normalization for assay standardization and increasedaccuracy.

In this example of throughput using arrays containing Raf 1 kinase inhibitor time coursetreated C32 cell lysates we tracked on the same slide total and phospho proteinsnormalized with GAPDH.

(Call out blue ERK and green S6, showing total protein and pERK stable, where pS6responding strongly over the time course.)

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Employing one of three channels for normalization with GAPDH provides a method foradjusting gross assay signal levels to compensate for variations in total deposited proteinsample during arraying, cell or tissue health or basic assay variability.

Measuring pERK inter-spot cell lysate signal consistencies across these slides wecompared different methods of normalization. Four methods included signalnormalization using measurements of either total protein or GAPDH from a separateslide, un-normalized data and lastly intra-spot multi-color multi-plexed normalizationwith GAPDH.

Intra-spot normalization provided the best data consistency, reducing %CV 10 – 25% overnon-normalized data and 5-15% over other external slide normalization methods. This isdue to normalization measurements performed on the exact same spot as the pERK asopposed to another separate slide.

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Fluorescent multi-color multi-plexing is not a new technique and LI-COR offers organicNear IR dyes for this application. Using the same Raf 1 kinase inhibitor time coursetreated C32 cell lysates arrays we compared our QNC with ArrayCAM method to theoptimized LI-COR method imaged with a two-laser confocal scanner.

The results showed the QNC and low-cost ArrayCAM provided equivalent results to themethods using the far more expensive and complex two-laser near IR scanner. In manycases, as seen here, we observed superior sensitivity with the QNC and ArrayCAM,yielding much greater resolution of these changes in protein expressions over these time-course studies.

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Yesterday we heard Ginny Espinosa present findings using two color QNC and ArrayCAMto measure intra-spot GAPDH normalized breast cancer pathway markers benchmarkedagainst amplified colorimetric detection systems using a slide auto-stainer.

We were interested to observe the absolute sensitivities between the QNC andcolorimetric methods so we compared LOD of GAPDH on a titration series of NIH 3T3 andJURKAT cell lysates between three experimental methods. QNC ArrayCAM methodperformed both on the autostainer at GMU and on the benchtop at Grace Bio-Labs andthe amplified colorimetric system at GMU. We found very similar LOD’s demonstratingQNC provides equivalent or better sensitivity without the need for expensive and timeconsuming amplification methods.

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We also observed sensitivity with low affinity primary antibody tests with QNC where a1:20 dilution was required to achieve equivalent or better LOD than the colorimetricresults on the auto-stainer. We felt this was a large amount of primary antibody andobserving experiments using manual bench-top methods and including agitation, wecould observe equivalent performance to the colorimetric and QNC 1:20 auto-stainermethods using a much lower 1:200 primary antibody dilution.

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For users who require the very large throughput of automated slide staining platforms,we’ve developed an amplification method and reagent set for the ArrayCAM systemwhich provides enhanced sensitivity using a better 1:200 primary antibody dilution fornon-optimal automated systems while still retaining the ability to multiplex detectionchannels.

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Typically, applications employing multi-color multiplexing require a combination ofdifferent species primary antibodies. RPPA requires stringent antibody specificity andvalidating different species antibodies can be difficult.

We have developed a method to perform two color multiplexing assays using samespecies primary antibodies incorporating antibody coupling to QNC with Protein G. Tosimplify this we have developed a one-step reagent kit for this application.

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Finally, we would like to take this opportunity to announce that we have begunproducing and offering TheraLin Tissue Fixative for Theranostics Health. For additionaldetails, you can check our website.

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