Odyssey Training. Dr. Christiane Kerscher

Transcription

1 Odyssey Training Dr. Christiane Kerscher

2 Odyssey CLx Imaging System Stable near-infrared fluorescence Detect multiple targets in the same lane Variety of assays and applications

3 Chemiluminescent Film Detection

4 Near-Infrared Fluorescent Detection

5 Stable Signals

6 Light Source and Sensitivity

7 Quantitative Western Blots R²=0.980 Protein Amount (ng)

8 Odyssey CLx Applications Western Blot Protein Gel In-Gel Western DNA Gel In-Cell Western Assay ELISA Protein Array EMSA Tissue Section Biodistribution Translational

9 Coomassie Protein Gel

10 Coomassie Protein Gel Computer Scanner (Silver Stain) Odyssey CLx Imaging System (Coomassie) Computer Scanner (Coomassie)

11 Coomassie Protein Gel Coomassie Odyssey CLx Silver Stain Computer Scanner Protein (µg) Protein (µg)

12 In-Gel Western

13 In-Gel Western Step 1: Perform electrophoresis Step 2: Cut away stacking gel Step 3: Fix proteins in gel Step 4: Incubate with primary and secondary antibodies Step 5: Image

14 In-Gel Western Transferrin T7-Tag Reprinted from Urh, M. et al. (2002) Poster presentation. Advances in Genome Biology and Technology Conference.

15 In-Cell Western Assay

16 In-Cell Western Assay A high-throughput, quantitative immunofluorescence assay performed in microplates in any well format

17 ICW Signaling Pathways Chen H, Kovar J, Sissons S, Cox K, Matter W, Chadwell F, et al. (2005). A Cell Based Immunocytochemical Assay For Monitoring Kinase Signaling Pathways And Drug Efficacy. Anal. Biochem.,

18 ICW sirna Knockdown Zhao Z et al. (2012) Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels. PLoS ONE. 7(3): e32628.

19 ICW Gene Expression Zhang Y-Y et al. (2014) Expression and Functional Characterization of NOD2 in Decidual Stromal Cells Isolated during the First Trimester of Pregnancy. PLoS ONE 9(6): e99612.

20 ICW Normalization Housekeeping Protein Cell Number CellTag 700 Stain (P/N ) Courtesy of Chad Dicky, Mayo Clinic Signaling Protein

21 On-Cell Western Assay

22 On-Cell Western Assay Cells are not permeabilized to detect only cell surface proteins

23 ELISA

24 ELISA FLISA NIR ELISA

25 EMSA

26 EMSA Step 1: Bind oligonucleotides to your sample Step 2: Perform non-denaturing electrophoresis Step 3: Image gel immediately without drying Step 3a: Run gel longer, if desired

27 Compare EMSA Sensitivity Radioactive Labeling Fluorescent Labeling Ying B-W, Fourmy D, Yoshizawa S. (2007). Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA. RNA. 13(11):

28 Protein Arrays

29 Adapted from Sheehan, KM et al. (2005) Use of Reverse Phase Protein Microarrays and Reference Standard Development for Molecular Network Analysis of Metastatic Ovarian Carcinoma. Mol Cell Proteomics. 4(4): Protein Arrays Step 1: Incubate protein samples with antibody array Step 2: Add biotinylated primary antibody and dye-labeled streptavidin Step 3: Image

30 Tissue Sections

31 Tissue Sections High Throughput Imaging of Brain Sections Kearn, CS (2004) Immunofluorescent Mapping of Cannibinoid CB1 and Dopamine D2 Receptors in the Mouse Brain. Lincoln: LI-COR Biosciences.

32 Tissue Sections Kearn, CS (2004) Immunofluorescent Mapping of Cannibinoid CB1 and Dopamine D2 Receptors in the Mouse Brain. Lincoln: LI-COR Biosciences.

33 Biodistribution and Organ Imaging

34 Biodistribution and Organ Imaging

35 Small Animal Optical Imaging

36 Small Animal Imaging in Near-Infrared

37 One Probe Simplifies Discovery IRDye Conjugated Probe in vitro Assays Clearance and in vivo Studies Biodistribution and Organ Imaging Tissue Section Imaging

38 Small Animal Imaging Workstation Evaluate binding activity Confirm specificity Verify binding capacity and clearance Validate histological specificity

39 Clinical Translation More than early phase clinical trials are either ongoing or completed with IRDye 800CW labeled monoclonal antibodies IRDye 700DX use with photoimmunotherapy phase I trial is ongoing

40 Western Blotting Hints/Tips

41 Western blot Protocol

42 SDS-PAGE

43 SDS-PAGE Sample loading buffer Run dye front off or cut off LI-COR 4X Protein Sample Loading Buffer P/N

44 SDS-PAGE Protein MW Standards 1/3 to 1/4 amount for current MW standard Odyssey Protein MW Marker LI-COR P/N

45 SDS-PAGE Sample loading Serial dilution of target Determine optimal loading & linear range

46 SDS-PAGE

47 Electrophoresis Pro-Tip: Check protein amount before gel loading Adjust volume with 1 x loading buffer and load equal volume per lane

48 Transfer Pro-Tip: Clean transfer tank prior to usage Soak transfer pads in 100% methanol for 10 minutes

49 Transfer Wet Tank Advantages Flexibility More complete transfer Favorable for a broader MW range Consistent antibody recognition Mini Trans-Blot System (Bio-Rad) Disadvantages Longer transfer times Cooling may be required

50 Transfer Semi-Dry Advantages Short transfer time Small buffer volume Mini Trans-Blot System (Bio-Rad) Disadvantages Low flexibility Variable transfer efficiency Choose the best method for your target!

51 Which Membrane to Choose PVDF Nitrocellulose Binding capacity ~ mg/cm 2 Physical strength, chemical resistance Requires wetting with methanol Binding capacity ~80mg/cm 2 Near instantaneous binding Supported is less brittle 0.45mm for proteins >20kDa 0.2mm for proteins <15kDa

52 PVDF 700 nm Channel 800 nm Channel Millipore Immobilon FL Millipore Immobilon P Bio-Rad Immun-Blot Pall BioTrace PVDF Perkin Elmer PolyScreen Amersham Hybond -P

53 Nitrocellulose 700 nm Channel 800 nm Channel Osmonics NitroBind Bio-Rad unsupported Odyssey Nitrocellulose

54 Take Your Research Further

55 Low Membrane Background in NIR Visible Fluorescence: High background NIR Fluorescence: Low background

56 Transfer Pro-Tip: Remove gel fragments Dry membrane (great for pausing!) Avoid writing on membranes Clean incubation boxes with methanol Normalize with REVERT Total Protein Stain

57 Normalization?

58 Normalization Total Protein REVERT Total Protein Stain P/N

59 Normalization Total Protein REVERT Total Protein Stain Western blot

60 Normalization Housekeeping Antibody P/N Rabbit anti-b Actin Mouse anti-b Actin Rabbit anti-b Tubulin Mouse anti-b Tubulin Rabbit anti-cox IV Contributed by Kimberly Wong, SUNY Upstate Medical Center, NY, United States

61 Normalization Signaling

62 Block

63 Blocker Selection 5% BSA Blocker 5% Milk Blocker Odyssey Blocking Buffer PKC-α

64 Blocker Selection Anti-AKT Block A Block B Block C Anti-ERK

65 Blocker Selection pakt AKT Blocking Buffer Optimization Kit LI-COR P/N

66 Secondary Detergents Tween % SDS % Blocking Primary PVDF Nitrocellulose

67 Block Pro-Tip: Use fresh blocking buffers Keep time/temperature consistent

68 Primary

69 Primary Antibody Selection Antibody Host Manufacturer Part # 1 α-gapdh Mouse Ambion GAPDH Sheep AbCam ab GAPDH Rabbit Rockland A33 4 GAPDH Mouse AbCam ab GAPDH Chicken ProSci Inc. XW GAPDH (N-14) Goat Santa Cruz Bio sc GAPDH (V-18) Goat Santa Cruz Bio sc α-gapdh Mouse Sigma G8795

70 Multiplexing L 3-sample comb Sample Sample Sample L P/N

71 Multiplexing Add two primary antibodies together Unrelated host species Check specificity

72 Secondary Detergents Tween % SDS % Blocking Primary PVDF Nitrocellulose

73 Primary Pro-Tip: Keep incubation time/temperature consistent Use fresh primary dilutions Wash with TBS or PBS + 0.1% Tween-20 Wash 4x 5 minutes don t over-wash!!

74 Secondary

75 Secondary Antibody 800 Channel Overlay 700 Channel NO CROSS ADSORPTION HIGHLY CROSS ADSORBED

76 Secondary Antibody Dilution Start at 1:20,000 Working Range 1:10,000 1:40,000

77 Secondary Detergents Tween % SDS % Blocking Primary PVDF Nitrocellulose

78 Secondary Pro-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!!

79 Image

80 Image Pro-Tip: Rinse membrane in TBS or PBS without Tween-20 Handle membranes with clean forceps Image wet or dry, test and be consistent Dry membranes for long term storage

81 Beyond blots

82 Quick Western Kit Quick Western Kit IRDye 680RD P/N

83 HRP & Odyssey Detection Chemi-IR Detection Kit P/N

84 HRP & Odyssey Detection Chemifluorescent Substrate P/N

85 Expand Your Research with IRDye Labeling Kits HS Click Chemistry

86 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 Services

87 Image Studio