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Odyssey Training Dr. Christiane Kerscher
Odyssey CLx Imaging System Stable near-infrared fluorescenceDetect multiple targets in the same laneVariety of assays and applications
Chemiluminescent Film Detection
Near-Infrared Fluorescent Detection
Stable Signals
Light Source and Sensitivity
Quantitative Western BlotsR² 0.980Protein Amount (ng)
Odyssey CLx ApplicationsWestern BlotProtein GelIn-Gel WesternIn-Cell Western AssayELISAProtein ArrayBiodistributionTranslationalTissue SectionDNA GelEMSA
Coomassie Protein Gel
Coomassie Protein GelComputer Scanner(Silver Stain)Odyssey CLxImaging System(Coomassie)Computer Scanner(Coomassie)
Coomassie Protein GelCoomassie – Odyssey CLxSilver Stain – Computer ScannerProtein (µg)Protein (µg)
In-Gel Western
In-Gel WesternStep 1: Perform electrophoresisStep 2: Cut away stacking gelStep 3: Fix proteins in gelStep 4: Incubate with primary andsecondary antibodiesStep 5: Image
In-Gel WesternTransferrinT7-TagReprinted from Urh, M. et al. (2002) Poster presentation. Advances in Genome Biology andTechnology Conference.
In-Cell WesternAssay
In-Cell Western Assay A high-throughput, quantitativeimmunofluorescence assay performed inmicroplates in any well format
ICW Signaling PathwaysChen H, Kovar J, Sissons S, Cox K, Matter W, Chadwell F, et al. (2005). A Cell BasedImmunocytochemical Assay For Monitoring Kinase Signaling Pathways And Drug Efficacy.Anal. Biochem., 136-142.
ICW siRNA KnockdownZhao Z et al. (2012) Increased Migration of Monocytes in Essential Hypertension IsAssociated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels.PLoS ONE. 7(3): e32628.
ICW Gene ExpressionZhang Y-Y et al. (2014) Expression and Functional Characterization of NOD2 in DecidualStromal Cells Isolated during the First Trimester of Pregnancy. PLoS ONE 9(6): e99612.
ICW NormalizationHousekeeping ProteinCourtesy of Chad Dicky, Mayo ClinicSignaling ProteinCell Number – CellTag 700 Stain(P/N 926-41090)
On-Cell Western Assay
On-Cell Western Assay Cells are not permeabilized to detect onlycell surface proteins
ELISA
ELISAFLISANIR ELISA
EMSA
EMSAStep 1: Bind oligonucleotides to your sampleStep 2: Perform non-denaturing electrophoresisStep 3: Image gel immediately without dryingStep 3a: Run gel longer, if desired
Compare EMSA SensitivityRadioactive LabelingFluorescent LabelingYing B-W, Fourmy D, Yoshizawa S. (2007). Substitution of the use of radioactivity byfluorescence for biochemical studies of RNA. RNA. 13(11):2042-2050.
Protein Arrays
Protein ArraysStep 1: Incubate protein samples withantibody arrayStep 2: Add biotinylated primaryantibody and dye-labeled streptavidinStep 3: ImageAdapted from Sheehan, KM et al. (2005) Use of Reverse Phase Protein Microarrays andReference Standard Development for Molecular Network Analysis of Metastatic OvarianCarcinoma. Mol Cell Proteomics. 4(4): 346-55.
Tissue Sections
Tissue SectionsHigh Throughput Imaging of Brain SectionsKearn, CS (2004) Immunofluorescent Mapping of Cannibinoid CB1 and Dopamine D2Receptors in the Mouse Brain. Lincoln: LI-COR Biosciences.
Tissue SectionsKearn, CS (2004) Immunofluorescent Mapping of Cannibinoid CB1 and Dopamine D2Receptors in the Mouse Brain. Lincoln: LI-COR Biosciences.
Biodistribution andOrgan Imaging
Biodistribution and Organ Imaging
Small AnimalOptical Imaging
Small Animal Imaging in Near-Infrared
One Probe Simplifies DiscoveryIRDye ConjugatedProbein vitroAssaysClearanceand in vivoStudiesBiodistributionand OrganImagingTissueSectionImaging
Small Animal Imaging Workstation Evaluate binding activityConfirm specificityVerify binding capacity and clearanceValidate histological specificity
Clinical Translation More than 1712 earlyphase clinical trials areeither ongoing orcompleted with IRDye 800CW labeledmonoclonal antibodies IRDye 700DX use withphotoimmunotherapyphase I trial is ongoing
Western Blotting Hints/Tips
Western blot Protocol
SDS-PAGE
SDS-PAGE Sample loading buffer Run dye front off or cut off LI-COR 4X Protein Sample Loading BufferP/N 928-40004
SDS-PAGE Protein MW Standards 1/3 to 1/4 amount for current MW standardOdyssey Protein MW MarkerLI-COR P/N 928-40000
SDS-PAGE Sample loading Serial dilution of target Determine optimal loading &linear range
SDS-PAGE
ElectrophoresisPro-Tip: Check protein amount before gel loading Adjust volume with 1 x loading buffer and load equal volume per lane
TransferPro-Tip: Clean transfer tank prior to usage Soak transfer pads in 100% methanol for 10 minutes
Transfer – Wet TankMini Trans-Blot System (Bio-Rad) Advantages Flexibility More complete transfer Favorable for a broader MW range Consistent antibody recognition Disadvantages Longer transfer times Cooling may be requiredhttp://biosupport.licor.com/docs/bt0609 LI-COR final Electrotransfer methods.pdf
Transfer – Semi-DryMini Trans-Blot System (Bio-Rad) Advantages Short transfer time Small buffer volume Disadvantages Low flexibility Variable transfer efficiencyChoose the best method for your target!
Which Membrane to ChoosePVDFNitrocellulose Binding capacity 150-160mg/cm2 Binding capacity 80mg/cm2 Physical strength, chemical resistance Near instantaneous binding Requires wetting with methanol Supported is less brittle0.45mm for proteins 20kDa0.2mm for proteins 15kDa
PVDF700 nm ChannelMillipore Immobilon FLMillipore Immobilon PBio-Rad Immun-Blot Pall BioTrace PVDFPerkin Elmer PolyScreen Amersham Hybond -P800 nm Channel
Nitrocellulose700 nm ChannelOsmonics NitroBindBio-Rad unsupportedOdyssey Nitrocellulose800 nm Channel
Take Your Research Further
Low Membrane Background in NIRVisible Fluorescence:High backgroundNIR Fluorescence:Low background
TransferPro-Tip: Remove gel fragments Dry membrane (great for pausing!) Avoid writing on membranes Clean incubation boxes with methanol Normalize with REVERT Total Protein Stain
Normalization?
Normalization – Total ProteinREVERT Total Protein StainP/N 926-11010
Normalization – Total ProteinREVERT TotalProtein StainWestern blot
Normalization – HousekeepingAntibodyP/NRabbit anti-b Actin926-42210Mouse anti-b Actin926-42212Rabbit anti-b Tubulin926-42211Mouse anti-b Tubulin926-42213Rabbit anti-COX IV926-42214Contributed by Kimberly Wong, SUNY Upstate Medical Center, NY, United States
Normalization – Signaling
Block
Blocker Selection5% BSA Blocker5% Milk BlockerOdyssey Blocking BufferPKC-α
Blocker SelectionAnti-AKTBlock AAnti-ERKBlock BBlock C
Blocker SelectionpAKTAKTBlocking Buffer Optimization KitLI-COR P/N 927-40040
ryPVDFNitrocelluloseSDS0.01–0.02%
BlockPro-Tip: Use fresh blocking buffers Keep time/temperature consistent
Primary
Primary Antibody Selection1 2 3 4 5 6 7 8Antibody12345678α-GAPDHGAPDHGAPDHGAPDHGAPDHGAPDH (N-14)GAPDH (V-18)α-GAPDHHostManufacturerPart CamRocklandAbCamProSci Inc.Santa Cruz BioSanta Cruz 6sc-20357G8795
Multiplexing3-sample combLSampleSampleSampleP/N 921-00000L
Multiplexing Add two primary antibodiestogether Unrelated host species Check specificity
ryPVDFNitrocelluloseSDS0.01–0.02%
PrimaryPro-Tip: Keep incubation time/temperatureconsistent Use fresh primary dilutions Wash with TBS or PBS 0.1% Tween-20 Wash 4x 5 minutes don’t over-wash!!
Secondary
800 Channel 700 ChannelOverlaySecondary AntibodyNO CROSS ADSORPTIONHIGHLY CROSS ADSORBED
Secondary Antibody DilutionStart at 1:20,000Working Range 1:10,000 – 1:40,000
ryPVDFNitrocelluloseSDS0.01–0.02%
SecondaryPro-Tip: IRDye 800CW for optimal sensitivity Incubate 1 hour at RT for best results Wash with TBS or PBS 0.1% Tween-20 Wash 4x 5 minutes don’t over-wash!!
Image
ImagePro-Tip: Rinse membrane in TBS or PBS withoutTween-20 Handle membranes with clean forceps Image wet or dry, test and be consistent Dry membranes for long term storage
Beyond blots
Quick Western KitQuick Western Kit – IRDye 680RDP/N 926-68100
HRP & Odyssey DetectionChemi-IR Detection KitP/N 926-32234
HRP & Odyssey DetectionChemifluorescent SubstrateP/N 928-30005
Expand Your Research with IRDyeLabeling KitsHSClick Chemistry
LI-COR Custom Services Dye Synthesis/cGMP Manufacturing Custom Small Scale Manufacturing Bioconjugation Organic Compound Synthesis Sample Analysis/Characterization Antibody Optimization Biological Protocol Development Biological Imaging Serviceswww.licor.com/customservices
Image Studio
Odyssey CLxApplications Western Blot In-Cell Western Assay Protein Gel In-Gel Western DNA Gel EMSA Tissue Section ELISA Protein Array Biodistribution Translational
