to 7c 1 ml sample obtained from cell culture on every hour was centrifuged at 10000 rpm at 4 u00b0 c for 10 minutes to remove the supernatant then whole cell sample was stored at u2212 80 u00b0 c for further analysis whole cell sample was suspended in 100 u03bcl sds page buffer 0 5m tris hcl ph 6 8 10 glycerol 5 sds 5 u03b2 mercaptoethanol 0 25 bromophenol blue and heated to 100 u00b0 c for 5 minutes the sample was loaded on 15 sds page and performed by electrophoresis to separate the proteins the proteins were detected by coomasie blue staining or silver staining bio rad usa the sample was loaded on 15 sds page electrophoresis was carried out and transferred onto nitrocellulose membrane at 15 v the mgfp 3a mutant on nitrocelluose membrane was detected using monoclonal antibody for histidine affinity ligand r amp d systems usa as the time passed after expression induction the expression pattern of recombinant mgfp 3a mutant adhesive protein was analyzed with sds page fig7 b unlike the other proteins of e coli the recombinant mgfp 3a mutant adhesive protein was detected as a heavy band with size of about 12 kda about 3 of proteins in total to investigate accurate band and expression pattern western.