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Exploitation of phage display technology has been well established in identification of protein-protein interactions and new bio-markers. We used lambda phage display technology to screen protein-protein interaction after expressing clones in bacterophage library. Bacteriophage cDNA library of specific cancer cell were obtained commercially and grown in E coli cultures. Large scale Libraries of cDNA were purified by using Lambda DNA Purification Kit (Cat#200391). Finally, specific protein-protein interactions were identified by phage lifting and detection with chemiluminescent method after probing membrane with HRP-conjugated protein.
Identification of protein-protein interaction by phage display method
Lambda phage cDNA library of cancer cells.
Bacteriophage cDNA library were obtained from ATCC (ATCC # 77426 FZ human large intestine (LII) cDNA library) and grown on Y1090 r - E.Coli host strain. Then lambda phage library were purified by using lambda DNA purification kit (cat #200391) titrated and PFU (plaque forming units)were determined by using x-gal as indicator. Phage infection of host strain were performed with 10000 pfu and screened for interaction and identification of proteins after transferring plaque product on to nitro cellulose membranes. Finally, detected with chemiluminiscence. Spots developed with desired protein as detection agent, were traced back from the master plate and reinoculated and purified with lambda DNA kit. Purified DNA is sequenced and corresponding gene is identified by NCBI BLAST.
We added 4 ml (Instead 3 ml, to get bigger plaques) of NZY top agar maintained at 45°C. (Using 48°C, didn't yield expected plaques formation)
We obtained high quality lambda DNA in a single experiment. We could able to demonstrate various protein-protein interactions as well as identification of new proteins which are specifically interacted with our interested protein. Results were reproducible after 18 months.
We stored phage particles at -80°C and reused as and when.
Very user friendly and yields high quality DNA from sequencing.
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The kit provides high quality DNA from sequencing. Also, the yield is very high and results were quite reproducible.
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