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Fig. 1 | Malaria Journal

Fig. 1

From: Self-assembling functional programmable protein array for studying protein–protein interactions in malaria parasites

Fig. 1

Outline of NAPPA protocol for detecting PPI in P. vivax. Step 1. Cloning: 35 DNA fragments from 20 P. vivax genes were amplified, adding attB recombination sequences for each one. Entry vector (pDONR221) recombination involved using BP clonase. Step 2. Sub-cloning: a second recombination involved positive clones (pDONR-Pv gene) and destination vectors (pANT7-cGST or pJFT7-nHalo) using LR clonase. Step 3. Printing and quality control: P. vivax plasmids were mixed with BSA, a crosslinker and rabbit polyclonal anti-GST antibody, and printed in spots on aminosilane-coated glass slides. After blocking, PicoGreen dye or IVTT (RRL or HCIVT) were used for staining slides. PicoGreen readings were used to calculate intra- and inter-slide correlation for assessing DNA printing quality. IVTT involved detecting captured proteins using mouse monoclonal anti-GST (primary) and HRP-linked anti-mouse IgG (secondary) antibodies, using tyramide as activated substrate. The amount of positive and negative spots was used to estimate expression performance. Step 4. Prey/bait co-expression: The IVTT system (RLL) was added together with prey plasmid to the array containing bait plasmid. Step 5. PPI detection: The PPI were revealed using mouse anti-Halo monoclonal (primary) and HRP-linked anti-mouse IgG (secondary) antibodies, using tyramide as activated substrate. Step 6. Data analysis: Fluorescence values were normalized to estimate positive signal values (greater than 1) for DNA printing and protein expression

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