Nucleic Acid Programmable Protein Array Workflow

Transcription

1 Dr. Sanjeeva Srivastava IIT Bombay Nucleic Acid Programmable Protein Array Workflow IIT Bombay 2 1

2 PISA Cell-free expression microarray NAPPA HaloTag Arrays MIST DAPA Figure 1 cdna (& GST tag) Capture antibody (e.g. anti-gst) GST Cell free expression GST Expressed protein BSA BS3 of target protein Ramachandran et al. Science 2004, 305, 86 Ramachandran et al. Nat Methods 2008, 5, 535 IIT Bombay 4 2

3 A repository of expression-ready clones in flexible cloning system that enables easy sub-cloning between various expression constructs A pipeline for quickly purifying DNA constructs and arraying them Robust printing chemistry HT capability at every step IIT Bombay 5 Array design and experimental planning Selection of surface and printing chemistries Culture expression construct clones Purification and concentration of DNA Re-suspension of expression construct DNA in array spotting buffer Array printing Quality control check IIT Bombay 6 3

4 Array planning Chemistry selection DNA purification Array printing IIT Bombay 7 IIT Bombay 4

5 Design experiment type & appropriate target protein test set Fit NAPPA chemistry utilized to assay being executed Decide array density & mapping Select robotic parameters for array execution IIT Bombay 9 IIT Bombay 10 5

6 6 x 6 spots per block IIT Bombay 11 IIT Bombay 6

7 IIT Bombay 13 Bioinformatics selection of target cdnas Primer design and PCR amplification Gel purification of PCR amplicons Cloning into entry construct Selection and culturing of isolated transformants IIT Bombay 14 7

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10 Glass Gold Nitrocellulose Hydrogel IIT Bombay 19 Silicon-based chemistry DNA can be attached covalently to slide by baking or UV irradiation Bake arrayed slides at 85 C UV cross-linking also provides good results IIT Bombay 20 10

11 Aminosilane coated slides contain high concentration of primary amino groups Amino group (+ charge) DNA backbone (- charge) IIT Bombay 21 Glass slides Acetone 99.9% Aminosilane (2% aminosilane solution in acetone) Metal 30-slide rack Rocking shaker IIT Bombay 22 11

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13 BSA BS 3 Printing master mix cdna with GST tag Antibody (anti- GST) IIT Bombay 25 BS3 cross-linker - water soluble, non cleavable, membrane impermeable Amino-reactive groups of N- hydroxysulfosuccinimide (Sulfo NHS), separated by spacer of 8C atoms Each protein contains a terminal amino group IIT Bombay 26 13

14 IIT Bombay 27 Environmental Humidity, temperature, vacuum deck Pin size/number Deck setup Plates, slides Array mapping Density, spot location Printing parameters Inking, stamping, print depth Pin washing IIT Bombay 28 14

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18 Spot size, shape & morphology Spot-to-spot consistency IIT Bombay 35 IIT Bombay 36 18

19 Precise liquid handling techniques Strict sample tracking throughout entire process Careful quality control of materials and reagents Maintain detailed description in notebooks Fidelity to protocols IIT Bombay 37 IIT Bombay 19

20 IIT Bombay 39 Printing batch 1 Printing batch 2 IIT Bombay 40 20

21 IIT Bombay IVTT mix Block Pri. Ab Sec. Ab TSA 30 ο C/90 min 15 ο C/30 min RT/ 45 min RT/ 45 min RT/ 10 min Blocking Expression Primary Antibody Secondary Antibody TSA Detection IIT Bombay 42 21

22 Anti-GST antibody IIT Bombay 43 Anti-p53 antibody IIT Bombay 44 22

23 Protein expression p53 expression IIT Bombay 45 DNA detection (PicoGreen) 46 Protein expression (Anti-GST antibody) Specific protein detection (Anti-p53 antibody) p53 expression (inset) 23

24 Day 1 printing Day 2 printing IIT Bombay 47 Spot-to-spot variation Slide-to-slide variation Day-to-day variation IIT Bombay 48 24

25 IIT Bombay 49 Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein, S. et al., Selfassembling protein mircoarrays. Science 2004, 305, Ramachandran N, Raphael JV, Hainsworth E, Demirkan G, Fuentes MG, Rolfs A, Hu Y, LaBaer J. Next-generation high-density self-assembling functional protein arrays. Nat Methods Jun;5(6): Harini Chandra, Sanjeeva Srivastava Cell-free synthesis-based protein microarrays and their applications. PROTEOMICS. Volume 10, Issue 4, pages , No. 4 February 2010 Harini Chandra, Panga Jaipal Reddy and Sanjeeva Srivastava Expert Review of Proteomics. Protein microarrays and novel detection platforms. February 2011, Vol. 8, No. 1, Pages /epr Joshua LaBaer, Niroshan Ramachandran Protein microarrays as tools for functional proteomics. Current Opinion in Chemical Biology. Volume 9, Issue 1, February 2005, Pages IIT Bombay 50 25

26 Drs. Joshua LaBaer and Niroshan Ramachandran, Ms. Eugenie Hainsworth, Harvard Institute of Proteomics, Harvard Medical School, USA CSHL, New York Proteomics course 2008 Dr. Kanad Baksi, DSS ImageTech, India IIT Bombay 26