f. 5 μl of water was then spotted on each spot for 30 sec and removed using Whatman filter paper strips. This step was repeated once. g. 1-2 μl of SAP matrix was then applied to each spot and allowed to dry. h. The chip was then placed in the SELDI machine

a. 5 μl of 10 mM HCl was added to each spot on the chip and removed after 5 min. using Whatman filter paper strips. b. Washing was given by spotting 3 μl of water for 30 sec on each spot followed by removal using Whatman filter paper strips. This step was repeated two times. c. 10 μl of low stringency/ high stringency buffer was then added to the spot and kept in humid chamber for 5 min. followed by removal using Whatman filter paper strips. d. 3 μl of sample prepared in low stringency/ high stringency buffer was then added to the spot and incubated in humid chamber for 30 min. e. Washed the spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH 7.0 for 30 sec and removed using Whatman filter paper strips. This step was repeated five times.

Fresh overnight culture of the E. coli strain (DH5α) was subcultured 1:100 in 250 ml LB/SOB media at 18 ̊C and 2500g and allowed to grow to an A600=0.55. Culture was chilled on ice and centrifuged at 2500g at 4 ̊C for 10 min. The cell pellet was redissolved in 80 ml ice-cold Inoue transformation buffer (55 mM MnCl2, 15 mM CaCl2, 250 mM KCl, 10 mM PIPES pH6.7). This cell suspension was centrifuged at 2500g at 4 ̊C for 10 min. and the cell pellet was resuspend in 20ml ice-cold Inoue buffer with 1.5 ml DMSO. This mixture was then placed on ice for 10 min. Aliquots of this suspension were dispensed into chilled, sterile microfuge tubes that were snap-frozen in a bath of liquid nitrogen. Tubes were stored at ─70 ̊C until required. For transformation the cells were thawed on ice and plasmid DNA was added followed by the standard transformation protocol