PDF Nucleic Acid Programmable Protein Array Workflow - NPTEL

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Dr. Sanjeeva Srivastava IIT Bombay

IIT Bombay Proteomics Course NPTEL

•  Nucleic Acid Programmable Protein Array

•  Workflow

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Figure 1

PISA NAPPA

MIST

DAPA HaloTag Arrays

microarray

IIT Bombay

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Proteomics Course NPTEL

(e.g. anti-GST) cDNA

Capture antibody

BSA BS3

GST

(& GST tag) Cell free expression of target protein

Expressed protein

GST

Ramachandran et al. Science 2004, 305, 86

Ramachandran et al. Nat Methods 2008, 5, 535

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•  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

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•  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

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Array printing

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•  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

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6 x 6 spots per block

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•  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

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Glass Gold Nitrocellulose Hydrogel

•  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

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•  Aminosilane coated slides contain high concentration of primary amino groups

•  Amino group (+ charge)

•  DNA backbone (- charge)

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•  Glass slides

•  Acetone 99.9%

•  Aminosilane (2% aminosilane solution in acetone)

•  Metal 30-slide rack

•  Rocking shaker

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BSA BS 3

cDNA with GST

tag

Antibody (anti-‐

GST)

Printing master mix

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•  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

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•  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

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  Spot size, shape &

morphology

•  Spot-to-spot consistency

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•  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

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Printing batch 1 Printing batch 2

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Blocking

IVTT mix 30

^ο

C/90 min 15

^ο

C/30 min

Expression

Sec.

Ab

Secondary Antibody RT/ 45 min Block

Primary Antibody RT/ 45 min

Pri.

Ab

TSA

Detection

TSA

RT/ 10 min

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Anti-p53 antibody

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Protein expression p53 expression

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DNA detection (PicoGreen)

Protein expression

(Anti-GST antibody) Specific protein detection

(Anti-p53 antibody) p53 expression

(inset)

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Day 1 printing Day 2 printing

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Spot-to-spot variation

Slide-to-slide variation

Day-to-day variation

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•  Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein, S. et al., Self- assembling protein mircoarrays. Science 2004, 305, 86–90.

•  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. 2008 Jun;5(6):535-8.

•  Harini Chandra, Sanjeeva Srivastava. 2010. Cell-free synthesis-based protein microarrays and their applications. PROTEOMICS. Volume 10, Issue 4, pages 717–730, No. 4 February 2010

•  Harini Chandra, Panga Jaipal Reddy and Sanjeeva Srivastava. 2011.

Expert Review of Proteomics. Protein microarrays and novel detection platforms. February 2011, Vol. 8, No. 1, Pages 61-79 10.1586/epr.10.99

•  Joshua LaBaer , Niroshan Ramachandran. 2005. Protein microarrays as tools for functional proteomics. Current Opinion in Chemical Biology.

Volume 9, Issue 1, February 2005, Pages 14–19.

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•  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

PDF Nucleic Acid Programmable Protein Array Workflow - NPTEL

Dr. Sanjeeva Srivastava IIT Bombay

Expressed protein

• Robust printing chemistry

• Array printing

• Decide array density & mapping

• Gel purification of PCR amplicons

• Silicon-based chemistry

• DNA backbone (- charge)

• BS3 cross-linker - water soluble, non cleavable, membrane impermeable

• Density, spot location

morphology

Printing batch 1 Printing batch 2

Protein expression p53 expression

• 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. 2008 Jun;5(6):535-8.

Volume 9, Issue 1, February 2005, Pages 14–19.

•  Robust printing chemistry

•  Array printing

•  Decide array density & mapping

•  Gel purification of PCR amplicons

•  Silicon-based chemistry

•  DNA backbone (- charge)

•  BS3 cross-linker - water soluble, non cleavable, membrane impermeable

•  Density, spot location

•  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. 2008 Jun;5(6):535-8.